Compositions and methods for preventing or treating diseases, conditions, or processes characterized by aberrant fibroblast proliferation and extracellular matrix deposition

ABSTRACT

The described invention provides compositions and methods for reducing progression of a fibrosis in a tissue of a subject selected from liver, kidney or vascular fibrosis, the progression of the fibrosis being characterized by aberrant fibroblast proliferation and extracellular matrix deposition in the tissue. The method includes administering a therapeutic amount of a pharmaceutical composition containing a polypeptide having the amino acid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or functional equivalent thereof, and a pharmaceutically acceptable carrier, wherein the therapeutic amount of the polypeptide is effective to reduce progression of the fibrosis, to treat remodeling of the tissue, or a combination thereof.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application claims the benefit of priority to U.S. ProvisionalApplication No. 62/080,784 filed Nov. 17, 2014, entitled “COMPOSITIONSAND METHODS FOR PREVENTING OR TREATING DISEASES, CONDITIONS, ORPROCESSES CHARACTERIZED BY ABERRANT FIBROBLAST PROLIFERATION ANDEXTRACELLULAR MATRIX DEPOSITION,” the content of which is incorporatedby reference herein in its entirety.

FIELD OF THE INVENTION

The invention is in the fields of cell and molecular biology,polypeptides, and therapeutic methods of use.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename: SEQLISTING.txt, daterecorded Nov. 17, 2015, file size 7354 bytes).

BACKGROUND

1. Mechanisms of Wound Healing and Fibrosis

The term “wound healing” refers to the process by which the body repairstrauma to any of its tissues, especially those caused by physical meansand with interruption of continuity.

A wound-healing response often is described as having three distinctphases-injury, inflammation and repair. Generally speaking, the bodyresponds to injury with an inflammatory response, which is crucial tomaintaining the health and integrity of an organism. If however it goesawry, it can result in tissue destruction.

Phase I: Injury

Injury caused by factors including, but not limited to, autoimmune orallergic reactions, environmental particulates, infection or mechanicaldamage often results in the disruption of normal tissue architecture,initiating a healing response. Damaged epithelial and endothelial cellsmust be replaced to maintain barrier function and integrity and preventblood loss, respectively. Acute damage to endothelial cells leads to therelease of inflammatory mediators and initiation of an anti-fibrinolyticcoagulation cascade, temporarily plugging the damaged vessel with aplatelet and fibrin-rich clot. For example, lung homogenates, epithelialcells or bronchoalveolar lavage fluid from idiopathic pulmonary fibrosis(IPF) patients contain greater levels of the platelet-differentiatingfactor, X-box-binding protein-1, compared with chronic obstructivepulmonary disease (COPD) and control patients, suggesting thatclot-forming responses are continuously activated. In addition, thrombin(a serine protease required to convert fibrinogen into fibrin) is alsoreadily detected within the lung and intra-alveolar spaces of severalpulmonary fibrotic conditions, further confirming the activation of theclotting pathway. Thrombin also can directly activate fibroblasts,increasing proliferation and promoting fibroblast differentiation intocollagen-producing myofibroblasts. Damage to the airway epithelium,specifically alveolar pneumocytes, can evoke a similar anti-fibrinolyticcascade and lead to interstitial edema, areas of acute inflammation andseparation of the epithelium from the basement membrane.

Platelet recruitment, degranulation and clot formation rapidly progressinto a phase of vasoconstriction with increased permeability, allowingthe extravasation (movement of white blood cells from the capillaries tothe tissues surrounding them) and direct recruitment of leukocytes tothe injured site. The basement membrane, which forms the extracellularmatrix underlying the epithelium and endothelium of parenchymal tissue,precludes direct access to the damaged tissue. To disrupt this physicalbarrier, zinc-dependent endopeptidases, also called matrixmetalloproteinases (MMPs), cleave one or more extracelluar matrixconstituents allowing extravasation of cells into, and out of, damagedsites. Specifically, MMP-2 (gelatinase A, Type N collagenase) and MMP-9(gelatinase B, Type IV collagenase) cleave type N collagens and gelatin,two important constituents of the basement membrane. Recent studies havefound that MMP-2 and MMP-9 are upregulated, highlighting thattissue-destructive and regenerative processes are common in fibroticconditions. The activities of MMPs are controlled by several mechanismsincluding transcriptional regulation, proenzyme regulation, and specifictissue inhibitors of MMPs. The balance between MMPs and the variousinhibitory mechanisms can regulate inflammation and determine the netamount of collagen deposited during the healing response.

Previous studies using a model of allergic airway inflammation andremodeling with MMP-2^(−/−), MMP-9^(−/−) and MMP-2^(−/−) MMP-9^(−/−)double knockout mice showed that MMP-2 and MMP-9 were required forsuccessful egression and clearance of inflammatory cells out of theinflamed tissue and into the airspaces. In the absence of these MMPs,cells were trapped within the parenchyma of the lung and were not ableto move into the airspaces, which resulted in fatal asphyxiation.

Phase II: Inflammation

Once access to the site of tissue damage has been achieved, chemokinegradients recruit inflammatory cells. Neutrophils, eosinophils,lymphocytes, and macrophages are observed at sites of acute injury withcell debris and areas of necrosis cleared by phagocytes.

The early recruitment of eosinophils, neutrophils, lymphocytes, andmacrophages providing inflammatory cytokines and chemokines cancontribute to local TGF-β and IL-13 accumulation. Following the initialinsult and wave of inflammatory cells, a late-stage recruitment ofinflammatory cells may assist in phagocytosis, in clearing cell debris,and in controlling excessive cellular proliferation, which together maycontribute to normal healing. Late-stage inflammation may serve ananti-fibrotic role and may be required for successful resolution ofwound-healing responses. For example, a late-phase inflammatory profilerich in phagocytic macrophages, assisting in fibroblast clearance, inaddition to IL-10-secreting regulatory T cells, suppressing localchemokine production and TGF-β, may prevent excessive fibroblastactivation.

The nature of the insult or causative agent often dictates the characterof the ensuing inflammatory response. For example, exogenous stimulilike pathogen-associated molecular patterns (PAMPs) are recognized bypathogen recognition receptors, such as toll-like receptors and NOD-likereceptors (cytoplasmic proteins that have a variety of functions inregulation of inflammatory and apoptotic responses), and influence theresponse of innate cells to invading pathogens. Endogenous dangersignals also can influence local innate cells and orchestrate theinflammatory cascade.

The nature of the inflammatory response dramatically influences residenttissue cells and the ensuing inflammatory cells. Inflammatory cellsthemselves also propagate further inflammation through the secretion ofchemokines, cytokines, and growth factors. Many cytokines are involvedthroughout a wound-healing and fibrotic response, with specific groupsof genes activated in various conditions. For example, chronic allergicairway disease in asthmatics is associated commonly with elevated type-2helper T cell (Th₂) related cytokine profiles (including, but notlimited to, interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6(IL-6), interleukin-13 (IL-13), and interleukin-9 (IL-9)), whereaschronic obstructive pulmonary disease and fibrotic lung disease (such asidiopathic pulmonary fibrosis) patients more frequently presentpro-inflammatory cytokine profiles (including, but not limited to,interleukin-1 alpha (IL-1α), interleukin-1 beta (IL-1β), interleukin-6(IL-6), tumor necrosis factor alpha (TNF-α), transforming growth factorbeta (TGF-β), and platelet-derived growth factors (PDGFs)). Each ofthese cytokines has been shown to exhibit significant pro-fibroticactivity, acting through the recruitment, activation and proliferationof fibroblasts, macrophages, and myofibroblasts.

Phase III: Tissue Repair and Contraction

The closing phase of wound healing consists of an orchestrated cellularre-organization guided by a fibrin (a fibrous protein that ispolymerized to form a “mesh” that forms a clot over a wound site)-richscaffold formation, wound contraction, closure and re-epithelialization.The vast majority of studies elucidating the processes involved in thisphase of wound repair have come from dermal wound studies and in vitrosystems.

Myofibroblast-derived collagens and smooth muscle actin (α-SMA) form theprovisional extracellular matrix, with macrophage, platelet, andfibroblast-derived fibronectin forming a fibrin scaffold. Collectively,these structures are commonly referred to as granulation tissues.Primary fibroblasts or alveolar macrophages isolated from idiopathicpulmonary fibrosis patients produce significantly more fibronectin andα-SMA than control fibroblasts, indicative of a state of heightenedfibroblast activation. It has been reported that IPF patients undergoingsteroid treatment had similar elevated levels of macrophage-derivedfibronectin as IPF patients without treatment. Thus, similar to steroidresistant IL-13-mediated myofibroblast differentiation,macrophage-derived fibronectin release also appears to be resistant tosteroid treatment, providing another reason why steroid treatment may beineffective. From animal models, fibronectin appears to be required forthe development of pulmonary fibrosis, as mice with a specific deletionof an extra type III domain of fibronectin (EDA) developed significantlyless fibrosis following bleomycin administration compared with theirwild-type counterparts.

In addition to fibronectin, the provisional extracellular matrixconsists of glycoproteins (such as PDGF), glycosaminoglycans (such ashyaluronic acid), proteoglycans and elastin. Growth factor andTGF-β-activated fibroblasts migrate along the extracellular matrixnetwork and repair the wound. Within skin wounds, TGF-β also induces acontractile response, regulating the orientation of collagen fibers.Fibroblast to myofibroblast differentiation, as discussed above, alsocreates stress fibers and the neo-expression of α-SMA, both of whichconfer the high contractile activity within myofibroblasts. Theattachment of myofibroblasts to the extracellular matrix at specializedsites called the “fibronexus” or “super mature focal adhesions” pull thewound together, reducing the size of the lesion during the contractionphase. The extent of extracellular matrix laid down and the quantity ofactivated myofibroblasts determines the amount of collagen deposition.To this end, the balance of matrix metalloproteinases (MMPs) to tissueinhibitor of metalloproteinases (TIMPs) and collagens to collagenasesvary throughout the response, shifting from pro-synthesis and increasedcollagen deposition towards a controlled balance, with no net increasein collagen. For successful wound healing, this balance often occurswhen fibroblasts undergo apoptosis, inflammation begins to subside, andgranulation tissue recedes, leaving a collagen-rich lesion. The removalof inflammatory cells, and especially α-SMA-positive myofibroblasts, isessential to terminate collagen deposition. Interestingly, in idiopathicpulmonary fibrosis patients, the removal of fibroblasts can be delayed,with cells resistant to apoptotic signals, despite the observation ofelevated levels of pro-apoptotic and FAS-signaling molecules. Thisrelative resistance to apoptosis may potentially underlie this fibroticdisease. However, several studies also have observed increased rates ofcollagen-secreting fibroblast and epithelial cell apoptosis inidiopathic pulmonary fibrosis, suggesting that yet another balancerequires monitoring of fibroblast apoptosis and fibroblastproliferation. From skin studies, re-epithelialization of the wound sitere-establishes the barrier function and allows encapsulated cellularre-organization. Several in vitro and in vivo models, using human or ratepithelial cells grown over a collagen matrix, or tracheal wounds invivo, have been used to identify significant stages of cell migration,proliferation, and cell spreading. Rapid and dynamic motility andproliferation, with epithelial restitution from the edges of the denudedarea occur within hours of the initial wound. In addition, slidingsheets of epithelial cells can migrate over the injured area assistingwound coverage. Several factors have been shown to regulatere-epithelialization, including serum-derived transforming growth factoralpha (TGF-α), and matrix metalloproteinase-7 (MMP-7) (which itself isregulated by TIMP-1).

Collectively, the degree of inflammation, angiogenesis, and amount ofextracellular matrix deposition all contribute to ultimate developmentof a fibrotic lesion. Thus, therapeutic intervention that interfereswith fibroblast activation, proliferation, or apoptosis requires athorough understanding and appreciation of all of the phases of woundrepair. Although these three phases are often presented sequentially,during chronic or repeated injury these processes function in parallel,placing significant demands on regulatory mechanisms. (Wilson and Wynn,Mucosal Immunol., 2009, 3(2):103-121).

2. Fibrosis as a Pathology

Fibrosis represents the formation or development of excess fibrousconnective tissue in an organ or tissue, which is formed as aconsequence of the normal or abnormal/reactive wound healing responseleading to a scar. Fibrosis is characterized by, for example, withoutlimitation, an aberrant deposition of an extracellular matrix protein,an aberrant promotion of fibroblast proliferation, an aberrant inductionof differentiation of a population of fibroblasts into a population ofmyofibroblasts, an aberrant promotion of attachment of myofibroblasts toan extracellular matrix, or a combination thereof.

Pro-Inflammatory Mediators

Accumulating evidence has suggested that polypeptide mediators known ascytokines, including various lymphokines, interleukins, and chemokines,are important stimuli to collagen deposition in fibrosis. Released byresident tissue cells and recruited inflammatory cells, cytokines arethought to stimulate fibroblast proliferation and increased synthesis ofextracellular matrix proteins, including collagen. For example, an earlyfeature in the pathogenesis of idiopathic pulmonary fibrosis is alveolarepithelial and/or capillary cell injury. This promotes recruitment intothe lung of circulating immune cells, such as monocytes, neutrophils,lymphocytes and eosinophils. These effector cells, together withresident lung cells, such as macrophages, alveolar epithelial andendothelial cells, then release cytokines, which stimulate target cells,typically fibroblasts, to replicate and synthesize increased amounts ofcollagen. Breakdown of extracellular matrix protein also may beinhibited, thereby contributing to the fibrotic process. (Coker andLaurent, Eur Respir J, 1998; 11:1218-1221)

Numerous cytokines have been implicated in the pathogenesis of fibrosis,including, without limitation, transforming growth factor-β (TGF-β),tumor necrosis factor-α (TNF-α), platelet-derived growth factor (PDGF),insulin-like growth factor-1 (IGF-1), endothelin-1 (ET-1) and theinterleukins, interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8(IL-8), and interleukin-17 (IL-17). Chemokine leukocytechemoattractants, including the factor Regulated upon Activation inNormal T-cells, Expressed and Secreted (RANTES), are also thought toplay an important role. Elevated levels of pro-inflammatory cytokines,such as Interleukin 8 (IL-8), as well as related downstream celladhesion molecules (CAMs) such as intercellular adhesion molecule-1(ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), matrixmetalloproteinases such as matrix metalloproteinase-7 (MMP-7), andsignaling molecules such as S100 calcium-binding protein A12 (S100A12,also known as calgranulin C), in the peripheral blood have been found tobe associated with mortality, lung transplant-free survival, and diseaseprogression in patients with idiopathic pulmonary fibrosis (Richards etal, Am J Respir Crit Care Med, 2012, 185: 67-76).

The TGF-β family of proteins has a potent stimulatory effect onextracellular matrix deposition, and in fact has been used inconstructing induced animal models of fibrosis through gene transfer. Invitro studies show that TGF-β1, secreted as a latent precursor, promotesfibroblast procollagen gene expression and protein synthesis. The datasuggest that the other mammalian isoforms, TGF-β2 and TGF-β3, alsostimulate human lung fibroblast collagen synthesis and reduce breakdownin vitro. In animal models of pulmonary fibrosis, enhanced TGF-β1 geneexpression is temporally and spatially related to increased collagengene expression and protein deposition. TGF-β1 antibodies reducecollagen deposition in murine bleomycin-induced lung fibrosis, and humanfibrotic lung tissue shows enhanced TGF-β1 gene and protein expression.

TNF-α can stimulate fibroblast replication and collagen synthesis invitro, and pulmonary TNF-α gene expression rises after administration ofbleomycin in mice. Soluble TNF-α receptors reduce lung fibrosis inmurine models, and pulmonary overexpression of TNF-α in transgenic miceis characterized by lung fibrosis. In patients with IPF or asbestosis (achronic inflammatory and fibrotic medical condition affecting theparenchymal tissue of the lungs caused by the inhalation and retentionof asbestos fibers), bronchoalveolar lavage fluid-derived macrophagesrelease increased amounts of TNF-α compared with controls.

Endothelin (ET-1) also fulfills the criteria for a profibrotic cytokine.This molecule promotes fibroblast proliferation and chemotaxis andstimulates procollagen production. It is present in the lungs ofpatients with pulmonary fibrosis, and a recent report suggests that theET-1 receptor antagonist, bosentan, ameliorates lung fibrosis whenadministered to experimental animals.

Unchecked Myofibroblast Proliferation/Activation and Fibrotic FociFormation

Differentiation of fibroblasts into myofibroblasts has long beenbelieved to be an important event in many conditions, including woundrepair and fibrosis. For example, it has been reported thatmyofibroblasts occur in areas of active fibrosis and are responsible forproduction and deposition of extracellular matrix (ECM) proteins inpulmonary fibrosis. (Liu, T. et al., Am J Respir Cell Mol Biol, 2007,37:507-517).

One hypothesis for the causation of idiopathic pulmonary fibrosissuggests that a still-unidentified stimulus produces repeated episodesof acute lung injury. Wound healing at these sites of injury ultimatelyleads to fibrosis, with loss of lung function. Fibroblast foci, thehallmark lesions of idiopathic pulmonary fibrosis, feature vigorousreplication of mesenchymal cells and exuberant deposition of freshextracellular matrix. Such foci are typical of alveolar epithelial-cellinjury, with endoluminal plasma exudation and collapse of the distal airspace. Mediators normally associated with wound healing, such astransforming growth factor-β1 (TGF-β1) and connective-tissue growthfactor, are expressed also at these sites. The driving force for thisfocal acute lung injury and wound repair is unknown. 3. Disease orConditions in which Fibrosis Plays a Role

Fibrosis has been implicated in a number of heterogeneous diseases orconditions, including, but not limited to, interstitial lung disease,such as idiopathic pulmonary fibrosis, acute lung injury (ALI),radiation-induced fibrosis, transplant rejection, liver fibrosis, renalfibrosis and vascular fibrosis.

3.1. Idiopathic Pulmonary Fibrosis (IPF)

Idiopathic Pulmonary fibrosis (IPF, also known as cryptogenic fibrosingalveolitis, CFA, or Idiopathic Fibrosing Interstitial Pneumonia) isdefined as a specific form of chronic, progressive fibrosinginterstitial pneumonia of uncertain etiology that occurs primarily inolder adults, is limited to the lungs, and is associated with theradiologic and histological pattern of usual interstitial pneumonia(UIP) (Raghu G. et al., Am J Respir Crit Care Med., 183(6):788-824,2011; Thannickal, V. et al., Proc Am Thorac Soc., 3(4):350-356, 2006).It may be characterized by abnormal and excessive deposition of fibrotictissue in the pulmonary interstitium. On high-resolution computedtomography (HRCT) images, UIP is characterized by the presence ofreticular opacities often associated with traction bronchiectasis. AsIPF progresses, honeycombing becomes more prominent (Neininger A. etal., J Biol Chem., 277(5):3065-8, 2002). Pulmonary function tests oftenreveal restrictive impairment and reduced diffusing capacity for carbonmonoxide (Thomas, T. et al., J Neurochem., 105(5): 2039-52, 2008).Studies have reported significant increases in TNF-α and IL-6 release inpatients with idiopathic pulmonary fibrosis (IPF) (Zhang, Y, et al. J.Immunol. 150(9):4188-4196, 1993), which has been attributed to the levelof expression of IL-1β (Kolb, M., et al. J. Clin. Invest,107(12):1529-1536, 2001). The onset of IPF symptoms, shortness of breathand cough, are usually insidious but gradually progress, with deathoccurring in 70% of patients within five years after diagnosis. Thisgrim prognosis is similar to numbers of annual deaths attributable tobreast cancer (Raghu G. et al., Am J Respir Crit Care Med.,183(6):788-824, 2011).

IPF afflicts nearly 130,000 patients in the United States, withapproximately 50,000 new patients annually and nearly 40,000 deaths eachyear worldwide (Raghu G. et al., Am J Respir Crit Care Med.,183(6):788-824, 2011). While these data are notable, a recent studyreported that IPF may be 5-10 times more prevalent than previouslythought, perhaps due to increasing prevalence or enhanced diagnosticcapabilities (Thannickal, V. et al., Proc Am Thorac Soc., 3(4):350-356,2006). Lung transplantation is considered a definitive therapy for IPF,but the five year survival post lung transplantation is less than 50%.Accordingly, even lung transplantation cannot be considered a “cure” forIPF. In addition to the physical and emotional toll on the patient, IPFis extremely expensive to treat and care for with national healthcarecosts to in the range of $2.8 billion dollars for every 100,000 patientsannually.

In addition, previous studies have suggested that superimposedenvironmental insults may be important in the pathogenesis of idiopathicpulmonary fibrosis. In most reported case series, up to 75 percent ofindex patients with idiopathic pulmonary fibrosis are current or formersmokers. In large epidemiologic studies, cigarette smoking has beenstrongly associated with idiopathic pulmonary fibrosis. In addition,many of the inflammatory features of idiopathic pulmonary fibrosis aremore strongly linked to smoking status than to the underlying lungdisease. Thus, cigarette smoking may be an independent risk factor foridiopathic pulmonary fibrosis. Latent viral infections, especially thoseof the herpes virus family, have also been reported to be associatedwith idiopathic pulmonary fibrosis.

Since there is no known effective treatment for IPF, including lungtransplantation, there remains a critical need for the development ofnovel therapeutics. There are a variety of therapeutic approachescurrently being investigated, including anti-fibrotic therapies that mayslow or inhibit the body's ability to produce scar or fibrotic tissueand pulmonary vasodilators to increase the tissue area for gas exchangein the lung. Aside from lung transplantation, potential IPF treatmentshave included corticosteroids, azathioprine, cyclophosphamide,anticoagulants, and N-acetylcysteine (Raghu G. et al., Am J Respir CritCare Med., 183(6):788-824, 2011). In addition, supportive therapies suchas oxygen therapy and pulmonary rehabilitation are employed routinely.However, none of these have definitely impacted the long term survivalof IPF patients, which further highlights the unmet medical need fortreatment options in IPF. As an example, despite mixed clinical programresults, InterMune's oral small-molecule Esbriet® (pirfenadone) receivedEuropean and Japanese approvals for patients with IPF. Esbriet® thusbecame the first medication specifically indicated for the treatment ofIPF; due to equivocal trial outcomes and drug side effects, the drug'sutility is viewed with skepticism in the United States, and did notreceive an FDA approval based on the data submitted at that time.Accordingly, a large phase 3 clinical trial is in progress to determineits efficacy to support a New Drug Application in the United States.

Histopathologically, IPF can be described as accumulation of activatedmyofibroblasts (or mesenchymal cells) in fibroblastic foci (Thannickal,V. et al., Proc Am Thorac Soc., 3(4):350-356, 2006). Impaired apoptosisof myofibroblasts may result in a persistent and dysregulated repairprocess that culminates in tissue fibrosis. Arguably, inflammation alsoplays a critical role in IPF, perhaps through cyclic acute stimulationof fibroblasts. These findings point to potential targets fortherapeutic intervention.

3.1.1. Pathogenesis of Idiopathic Pulmonary Fibrosis (IPF)

While pathogenic mechanisms are incompletely understood, the currentlyaccepted paradigm proposes that injury to the alveolar epithelium isfollowed by a burst of pro-inflammatory and fibroproliferative mediatorsthat invoke responses associated with normal tissue repair. For unclearreasons, these repair processes never resolve and progressive fibrosisensues. (Selman M, et al., Ann Intern Med, 134(2):136-151, 2001; Noble,P. and Homer R., Clin Chest Med, 25(4):749-58, 2004; Strieter, R.,Chest, 128 (5 Suppl 1):526S-532S, 2005).

3.1.2. Bleomycin Mouse Model of Pulmonary Fibrosis

Although a number of animal models exist and can be useful (e.g., theTGF-β adenovirus transduction model or the radiation-induced fibrosismodel), the bleomycin model is well-documented and the bestcharacterized murine model in use today to demonstrate efficacy of aparticular drug or protein kinase inhibitor in thepost-inflammatory/pre-fibrotic/fibro-preventive stages (Vittal, R. etal., J Pharmacol Exp Ther., 321(1):35-44, 2007; Vittal, R. et al., Am JPathol., 166(2):367-75, 2005; Hecker L. et al., Nat Med., 15(9):1077-81,2009).

The antibiotic bleomycin was originally isolated from Streptomycesverticillatus (Umezawa, H. et al., Cancer 20: 891-895, 1967). Thisantibiotic was subsequently found to be effective against squamous cellcarcinomas and skin tumors (Umezawa, H., Fed Proc, 33: 2296-2302, 1974);however, its usefulness as an anti-neoplastic agent was limited bydose-dependent pulmonary toxicity resulting in fibrosis (Muggia, F. etal., Cancer Treat Rev, 10: 221-243, 1983). The delivery of bleomycin viathe intratracheal route (generally 1.25-4 U/kg, depending on the source)has the advantage that a single injection of the drug produces lunginjury and resultant fibrosis in rodents (Phan, S. et al., Am Rev RespirDis 121: 501-506, 1980; Snider, G. et al., Am Rev Respir Dis. 117:289-297, 1978; Thrall, R. et al., Am J Pathol, 95: 117-130, 1979).Intratracheal delivery of the drug to rodents results in direct damageinitially to alveolar epithelial cells. This event is followed by thedevelopment of neutrophilic and lymphocytic pan-alveolitis within thefirst week (Janick-Buckner, D. et al., Toxicol Appl Pharmacol.,100(3):465-73, 1989). Subsequently, alveolar inflammatory cells arecleared, fibroblast proliferation is noted, and extracellular matrix issynthesized (Schrier D. et al., Am Rev Respir Dis., 127(1):63-6, 1983).The development of fibrosis in this model can be seen biochemically andhistologically by day 14 with maximal responses generally noted arounddays 21-28 (Izbicki G. et al., Int J Exp Pathol., 83(3):111-9, 2002;Phan, S. et al., Chest., 83(5 Suppl):44S-45S, 1983). Beyond 28 days,however, the response to bleomycin is more variable. Original reportssuggest that bleomycin delivered intratracheally may induce fibrosisthat progresses or persists for 60-90 days (Thrall R. et al., Am JPathol., 95(1):117-30, 1979; Goldstein R., et al., Am Rev Respir Dis.,120(1):67-73, 1979; Starcher B. et al., Am Rev Respir Dis.,117(2):299-305, 1978); however, other reports demonstrate aself-limiting response that begins to resolve after this period (ThrallR. et al., Am J Pathol., 95(1):117-30, 1979; Phan, S. et al., Chest,83(5 Suppl): 44S-45S, 1983; Lawson W. et al., Am J Pathol. 2005;167(5):1267-1277). While the resolving nature of this model does notmimic human disease, this aspect of the model offers an opportunity forstudying fibrotic resolution at these later time points.

3.2. Acute Lung Injury (ALI)

Acute lung injury (ALI) and its more severe form, the acute respiratorydistress syndrome (ARDS), are syndromes of acute respiratory failurethat result from acute pulmonary edema and inflammation. ALI/ARDS is acause of acute respiratory failure that develops in patients of all agesfrom a variety of clinical disorders, including sepsis (pulmonary andnonpulmonary), pneumonia (bacterial, viral, and fungal), aspiration ofgastric and oropharyngeal contents, major trauma, and several otherclinical disorders, including severe acute pancreatitis, drug over dose,and blood products (Ware, L. and Matthay, M., N Engl J Med,342:1334-1349, 2000). Most patients require assisted ventilation withpositive pressure. The primary physiologic abnormalities are severearterial hypoxemia as well as a marked increase in minute ventilationsecondary to a sharp increase in pulmonary dead space fraction. Patientswith ALI/ARDS develop protein-rich pulmonary edema resulting fromexudation of fluid into the interstitial and airspace compartments ofthe lung secondary to increased permeability of the barrier. Additionalpathologic changes indicate that the mechanisms involved in lung edemaare complex and that edema is only one of the pathophysiologic events inALI/ARDS. One physiologic consequence is a significant decrease in lungcompliance that results in an increased work of breathing (Nuckton T. etal., N Engl J Med, 346:1281-1286, 2002), one of the reasons why assistedventilation is required to support most patients.

It was suggested that mechanical ventilation (MV), a mainstay treatmentfor ALI, potentially contributes to and worsens permeability by exactingmechanical stress on various components of the respiratory systemcausing ventilator-associated lung injury (VALI) (Fan, E. et al., JAMA,294:2889-2896, 2005; MacIntyre N., Chest, 128:561S-567, 2005). A recenttrial demonstrated a significant improvement in survival in patientsventilated with low (LV_(T)) compared to high tidal volumes (HV_(T))(The Acute Respiratory Distress Syndrome N. Ventilation with Lower TidalVolumes as Compared with Traditional Tidal Volumes for Acute Lung Injuryand the Acute Respiratory Distress Syndrome. N Engl J Med;342:1301-1308, 2000). Other than ventilating at lower tidal volumes,which presumably imparts lower mechanical stress, there is littlemechanistic understanding of the pathophysiology and no directedtherapies for VALI.

It was suggested that the high tidal volumes (HV_(T)) mechanicalventilation (MV) results in phosphorylation of p38 MAP kinase,activation of MK2, and phosphorylation of HSPB1, a process that causesactin to disassociate from HSPB1 and polymerize to form stress fibers,which ultimately leads to paracellular gaps and increased vascularpermeability. Furthermore, it was shown that inhibiting p38 MAP kinaseor its downstream effector MK2 prevents the phosphorylation of HSPB1 andprotects from vascular permeability by abrogating actin stress fiberformation and cytoskeletal rearrangement, suggesting that targetedinhibition of MK2 could be a potential therapeutic strategy for thetreatment of acute lung injury (Damarla, M. et al., PLoS ONE, 4(2):E4600, 2009).

Moreover, studies have suggested that pulmonary fibrosis can also resultfrom ALI. ALI may completely resolve or proceed to fibrosing alveolitisaccompanied by persistent low oxygen in the blood (hypoxemia) and areduced ability of the lung to expand with every breath (reducedpulmonary compliance). It was suggested that while the etiology ofinjury-induced lung fibrosis is different from idiopathic pulmonaryfibrosis, both diseases share a common pathological mechanism, i.e.,infiltration of fibroblasts into the airspaces of lung (Tager et al.,Nat. Med. 14: 45-54, 2008; Ley, K. and Zarbock, A., Nat. Med. 14: 20-21;2008).

3.3. Radiation-Induced Fibrosis

Fibrosis is a common sequela of both cancer treatment by radiotherapyand accidental irradiation. Fibrotic lesions following radiotherapy havebeen described in many tissues, including skin (Bentzen, S. et al.,Radiother. Oncol. 15: 261-214, 1989; Brocheriou, C., et al., Br. J.Radiol. Suppl. 19: 101-108, 1986), lung (Lopez Cardozo, B. et al., Int.J. Radiat. Oncol. Biol. Phys., 11: 907-914, 1985), heart (Fajardo, L.and Stewart, J., Lab. Invest., 29: 244-257, 1973), and liver (Ingold, J.et al., Am. J. Roentgenol., 93: 200-208, 1965).

In the lung (late responding tissue), two radiation toxicity syndromes,radiation pneumonitis and pulmonary fibrosis, may occur. Pneumonitis ismanifested 2-3 months after radiotherapy is completed. Pathologically,pneumonitis is characterized by interstitial edema, the presence ofinterstitial and alveolar inflammatory cells, and an increase in thenumber of type II pneumocytes (Gross, N. et al., Radiat. Res., III:143-50, 1981; Guerry-Force, M. et al., Radiat. Res. 114: 138-53, 1988).In pneumonitis, the primary damage to the tissue is most likely causedby depletion of parenchymal cells (Hendry, J., Radiat. Oncol. Vol. 4, 2:123-132, 1994; Rosiello, R. et al., Am. Rev. Respir. Dis., 148:1671-1676, 1993; Travis, E. and Terry, N., Front. Radiat. Ther. Oncol.,23: 41-59, 1989).

The fibrotic reaction is typified by increased interstitial collagendeposition, thickening of vascular walls and vascular occlusions(Vergava, J. et al., Int. J. Radiat. Oncol. Biol. Phys. 2: 723-732,1987). Histological examinations of fibrotic lesions have revealed thatfibrotic tissue contains infiltrating inflammatory cells, fibroblasts,and larger amounts of various extracellular matrix components. Infibrotic tissues, an enhanced synthesis and deposition of theinterstitial collagens, fibronectin, and proteoglycans have beendescribed (Maasiha, P. et al., Int. J. Radiat. Oncol. Biol. Phys. 20:973-980, 1991), and this has been interpreted as the result of theradiation-induced modulation of the fibroblast cell system (Remy, J. etal., Radiat. Res. 125: 14-19, 1991).

Radiation-induced fibrosis, especially of the lung, was suggested to bedue to an interplay of cellular and molecular events between severalcell systems engaged in a fibrotic reaction. Irradiation alone is ableto induce a premature terminal differentiation process of thefibroblast/fibrocyte cell system resulting in the enhanced accumulationof postmitotic fibrocytes, which are characterized by a several-foldincrease in the synthesis of interstitial collagens. Concomitantly,irradiation of accompanying parenchymal cell types, such as alveolarmacrophages and alveolar type II pneumocytes, induces the immediatesynthesis of specific cytokines, like TGF-β1, which then alter theinteraction of the parenchymal cells with the fibroblast cell system.TGF-β1, as one of the major cytokines responsible for the fibroticreaction, induces the fibroblast proliferation via an expansion of theprogenitor fibroblast cell types as well as a premature terminaldifferentiation of progenitor fibroblasts into post-mitotic fibrocytes.This leads to an accumulation of post-mitotic fibrocytes due to adisturbance of the well-balanced cell type ratio of progenitorfibroblasts and post-mitotic fibrocytes. It was proposed that thepathophysiological tissue response following irradiation is caused by analtered cytokine- and growth factor-mediated interaction ofmulticellular cell systems resulting in the disturbance of thewell-balanced cell type ratio of the interstitial fibroblast/fibrocytecell system. (Rodemann, H. and Bamberg, M., Radiotherapy and Oncology,35, 83-90, 1995).

3.4. Transplant Rejection

Transplantation is the act of transferring cells, tissues, or organsfrom one site to another. The malfunction of an organ system can becorrected with transplantation of an organ (e.g., kidney, liver, heart,lung, or pancreas) from a donor. However, the immune system remains themost formidable barrier to transplantation as a routine medicaltreatment, and rejection of such organ often corresponds to a fibroticphenotype in the grafted organ. The immune system has developedelaborate and effective mechanisms to combat foreign agents. Thesemechanisms are also involved in the rejection of transplanted organs,which are recognized as foreign by the host's immune system.

The degree of immune response to a graft depends partly on the degree ofgenetic disparity between the grafted organ and the host. Xenografts,which are grafts between members of different species, have the mostdisparity and elicit the maximal immune response, undergoing rapidrejection. Autografts, which are grafts from one part of the body toanother (e.g., skin grafts), are not foreign tissue and, therefore, donot elicit rejection. Isografts, which are grafts between geneticallyidentical individuals (e.g., monozygotic twins), also undergo norejection.

Allografts are grafts between members of the same species that differgenetically. This is the most common form of transplantation. The degreeto which allografts undergo rejection depends partly on the degree ofsimilarity or histocompatibility between the donor and the host.

The degree and type of response also vary with the type of thetransplant. Some sites, such as the eye and the brain, areimmunologically privileged (i.e., they have minimal or no immune systemcells and can tolerate even mismatched grafts). Skin grafts are notinitially vascularized and so do not manifest rejection until the bloodsupply develops. The lungs, heart, kidneys, and liver are highlyvascular organs and often lead to a vigorous cell mediated response inthe host, requiring immunosuppressive therapies.

Constrictive bronchiolitis (CB), also termed in lung transplant patientsobliterative bronchiolitis, is inflammation and fibrosis occurringpredominantly in the walls and contiguous tissues of membranous andrespiratory bronchioles with resultant narrowing of their lumens. CB isfound in a variety of settings, most often as a complication of lung andheart-lung transplantation (affecting 34% to 39% of patients, usually inthe first 2 years after transplantation) and bone marrowtransplantation, but also in rheumatoid arthritis, after inhalation oftoxic agents such as nitrogen dioxide, after ingestion of certain drugssuch as penicillamine and ingestion of the East Asian vegetable Sauropusandrogynous, and as a rare complication of adenovirus, influenza type A,measles, and Mycoplasma pneumoniae infections in children. In lungtransplants, CB is the single most important factor leading to deaththereafter. In one study, the overall mortality rate was 25%. However,at the same time, 87% of patients who were asymptomatic and diagnosedsolely by transbronchial biopsy had resolution or stabilization ofdisease. Decreases in FEV₁ from baseline can be used to clinicallysupport CB in transplant patients; the term bronchiolitis obliteranssyndrome is used to denote this clinical dysfunction, and a gradingsystem has been established for it that is now widely used in theliterature. Significant risk factors for the development of CB in lungtransplants include alloantigen-dependent and -independent mechanisms.In the former group are late acute rejection and HLA mismatches at the Aloci; in the latter are ischemia/reperfusion injuries to airways thatresult from the transplantation surgery and cytomegalovirus infection(Schlesinger C. et al, Curr Opin Pulm. Med., 4(5): 288-93, 1998).

Mechanisms of Rejection

The immune response to a transplanted organ consists of both cellular(lymphocyte mediated) and humoral (antibody mediated) mechanisms.Although other cell types are also involved, the T cells are central inthe rejection of grafts. The rejection reaction consists of thesensitization stage and the effector stage.

Sensitization Stage

In this stage, the CD4 and CD8 T cells, via their T-cell receptors,recognize the alloantigens expressed on the cells of the foreign graft.Two signals are needed for recognition of an antigen; the first isprovided by the interaction of the T cell receptor with the antigenpresented by MHC molecules, the second by a co-stimulatoryreceptor/ligand interaction on the T cell/APC surface. Of the numerousco-stimulatory pathways, the interaction of CD28 on the T cell surfacewith its APC surface ligands, B7-1 or B7-2 (commonly known as CD80 orCD86, respectively), has been studied the most (Clarkson, M. and Sayegh,M., Transplantation; 80(5): 555-563, 2005). In addition, cytotoxic Tlymphocyte-associated antigen-4 (CTLA4) also binds to these ligands andprovides an inhibitory signal. Other co-stimulatory molecules includeCD40 and its ligand CD40L (CD154). Typically, helices of the MHCmolecules form the peptide-binding groove and are occupied by peptidesderived from normal cellular proteins. Thymic or central tolerancemechanisms (clonal deletion) and peripheral tolerance mechanisms (e.g.,anergy) ensure that these self-peptide MHC complexes are not recognizedby the T cells, thereby preventing autoimmune responses.

Effector Stage

Alloantigen-dependent and independent factors contribute to the effectormechanisms. Initially, nonimmunologic “injury responses” (ischemia)induce a nonspecific inflammatory response. Because of this, antigenpresentation to T cells is increased as the expression of adhesionmolecules, class II MHC, chemokines, and cytokines is upregulated. Italso promotes the shedding of intact, soluble MHC molecules that mayactivate the indirect allorecognition pathway. After activation,CD4-positive T cells initiate macrophage-mediated delayed typehypersensitivity (DTH) responses and provide help to B cells forantibody production.

Various T cells and T cell-derived cytokines such as IL-2 and IFN-γ areupregulated early after transplantation. Later, β-chemokines like RANTES(regulated upon activation, normal T cell expressed and secreted),IP-10, and MCP-1 are expressed, and this promotes intense macrophageinfiltration of the allograft. IL-6, TNF-α, inducible nitric oxidesynthase (iNOS) and growth factors, also play a role in this process.Growth factors, including TGF-β and endothelin, cause smooth muscleproliferation, intimal thickening, interstitial fibrosis, and, in thecase of the kidney, glomerulosclerosis.

Endothelial cells activated by T cell-derived cytokines and macrophagesexpress class II MHC, adhesion molecules, and co-stimulatory molecules.These can present antigen and thereby recruit more T cells, amplifyingthe rejection process. CD8-positive T cells mediate cell-mediatedcytotoxicity reactions either by delivering a “lethal hit” or,alternatively, by inducing apoptosis.

In addition, emerging studies have suggested involvement of fibroticprocesses in chronic transplant rejection of an organ transplant. Forexample, it was shown that chronic lung allograft rejection is mediatedby a relative deficiency of allograft endothelial cell-derived HIF-1α,leading to fibrotic remodeling of the transplanted organ (Wilkes, D., JClin Invest., 121(6): 2155-2157, 2011; Jiang, X. et al., J Clin Invest.,121(6): 2336-2349, 2011).

3.5. Chronic Obstructive Pulmonary Disease (COPD)

Chronic obstructive pulmonary disease (COPD) is a collective descriptionfor lung diseases represented by chronic and relatively irreversibleexpiratory airflow dysfunction due to some combination of chronicobstructive bronchitis, emphysema, and/or chronic asthma. COPD is causedby a range of environmental and genetic risk factors, including smokingthat contributes to the disease.

The prevalence of COPD is increasing worldwide, and COPD has become thefourth leading cause of death in the United States. In the UnitedStates, despite the decrease in cigarette smoking in recent decades,both the prevalence of, and the mortality associated with, COPD haveincreased and are projected to continue to increase for some years yet.Furthermore, COPD is costly, and acute exacerbations, which occurroughly once a year in patients with COPD of moderate or greaterseverity, constitute the most expensive component.

In COPD, airflow obstruction can occur on the basis of either of twovery different pathophysiological processes in the lung: 1) inflammationof the parenchyma resulting in proteolysis of the lung parenchyma andloss of lung elasticity (emphysema); and 2) inflammation, scarring andnarrowing of the small airways (“small airway disease”). In anindividual patient, one of these processes, which may be controlled bydifferent genetic factors, may predominate although both usuallyco-exist. Ultimately, both of these processes produce similar patternsof functional impairment: decreased expiratory flow, hyperinflation andabnormalities of gas exchange.

At an early stage of COPD, the following symptoms are found in the lungsof COPD patients: 1) breach of airway epithelium by damaging aerosols,2) accumulation of inflammatory mucous exudates, 3) infiltration of theairway wall by inflammatory immune cells, 4) airwayremodeling/thickening of the airway wall and encroachment on lumenalspace, and 5) increased resistance to airflow. During this early stage,smooth muscle contraction and hyper-responsiveness also increaseresistance, but the increased resistance is relieved by bronchodilators.

At an advanced stage, COPD patients characteristically developdeposition of fibrous connective tissue in the subepithelial andaventitial compartments surrounding the airway wall. Suchperibronchiolar fibrosis contributes to fixed airway obstruction byrestricting the enlargement of airway caliber that occurs with lunginflation.

3.5.1. Chronic Bronchitis

Chronic bronchitis is defined as the presence of chronic cough andsputum production for at least three months of two consecutive years inthe absence of other diseases recognized to cause sputum production. Inchronic bronchitis, epidemiologically the bronchial epithelium becomeschronically inflamed with hypertrophy of the mucus glands and anincreased number of goblet cells. The cilia are also destroyed and theefficiency of the mucociliary escalator is greatly impaired. Mucusviscosity and mucus production are increased, leading to difficulty inexpectorating. Pooling of the mucus leads to increased susceptibility toinfection.

Microscopically there is infiltration of the airway walls withinflammatory cells. Inflammation is followed by scarring and remodelingthat thickens the walls and also results in narrowing of the airways. Aschronic bronchitis progresses, there is squamous metaplasia (an abnormalchange in the tissue lining the inside of the airway) and fibrosis(further thickening and scarring of the airway wall). The consequence ofthese changes is a limitation of airflow. Repeated infections andinflammation over time leads to irreversible structural damage to thewalls of the airways and to scarring, with narrowing and distortion ofthe smaller peripheral airways.

3.5.2. Emphysema

Emphysema is defined in terms of its pathological features,characterized by abnormal dilatation of the terminal air spaces distalto the terminal bronchioles, with destruction of their wall and loss oflung elasticity. Bullae (blisters larger than 1 cm wide) may develop asa result of overdistention if areas of emphysema are larger than 1 cm indiameter. The distribution of the abnormal air spaces allows for theclassification of the two main patterns of emphysema: panacinar(panlobular) emphysema, which results in distension, and destruction ofthe whole of the acinus, particularly the lower half of the lungs.Centriacinar (centrilobular) emphysema involves damage around therespiratory bronchioles affecting the upper lobes and upper parts of thelower lobes of the lung. Certain forms of emphysema are furthermoreknown to be associated with fibrosis.

The destructive process of emphysema is predominantly associated withcigarette smoking. Cigarette smoke is an irritant and results inlow-grade inflammation of the airways and alveoli. It is known thatcigarettes contain over 4,000 toxic chemicals, which affect the balancebetween the antiprotease and proteases within the lungs, causingpermanent damage. Inflammatory cells (macrophages and neutrophils)produce a proteolytic enzyme known as elastase, which destroys elastin,an important component of lung tissue.

The alveoli or air sacs of the lung contain elastic tissue, whichsupports and maintains the potency of the intrapulmonary airways. Thedestruction of the alveolar walls allows narrowing in the small airwaysby loosening the guy ropes that help keep the airways open. Duringnormal inspiration, the diaphragm moves downwards while the rib cagemoves outwards, and air is drawn into the lungs by the negative pressurethat is created. On expiration, as the rib cage and diaphragm relax, theelastic recoil of the lung parenchyma pushes air upwards and outwards.With destruction of the lung parenchyma, which results in floppy lungsand loss of the alveolar guy ropes, the small airways collapse and airtrapping occurs, leading to hyperinflation of the lungs. Hyperinflationflattens the diaphragm, which results in less effective contraction andreduced alveolar efficiency, which in turn leads to further airtrapping. Over time the described mechanism leads to severe airflowobstruction, resulting in insufficient expiration to allow the lungs todeflate fully prior to the next inspiration.

3.5.3 Chronic Asthma

Asthma is defined as a chronic inflammatory condition of the airways,leading to widespread and variable airways obstruction that isreversible spontaneously or with treatment. In some patients withchronic asthma, the disease progresses, leading to irreversible airwayobstruction, particularly if the asthma is untreated, either because ithas not been diagnosed or mismanaged, or if it is particularly severe.Children with asthma have a one in ten chance of developing irreversibleasthma, while the risk for adult-onset asthmatics is one in four.Studies also have found that in both children and adults that asthmamight lead to irreversible deterioration in lung function if theirasthma was not treated appropriately, particularly with corticosteroidtherapy.

The airway inflammation in asthma over time can lead to remodeling ofthe airways through increased smooth muscle, disruption of the surfaceepithelium increased collagen deposition and thickening of the basementmembrane.

3.6 Other Types of Fibrosis

Other types of fibrosis include, without limitation, cystic fibrosis ofthe pancreas and lungs, injection fibrosis, endomyocardial fibrosis,mediastinal fibrosis, myelofibrosis, retroperitoneal fibrosis, andnephrogenic systemic fibrosis.

Cystic fibrosis (CF, mucovidosis, mucovisidosis) is an inheritedautosomal recessive disorder. It is one of the most common fatal geneticdisorders in the United States, affecting about 30,000 individuals, andis most prevalent in the Caucasian population, occurring in one of every3,300 live births. The gene involved in cystic fibrosis, which wasidentified in 1989, codes for a protein called the cystic fibrosistransmembrane conductance regulator (CFTR). CFTR normally is expressedby exocrine epithelia throughout the body and regulates the movement ofchloride ions, bicarbonate ions and glutathione into and out of cells.In cystic fibrosis patients, mutations in the CFTR gene lead toalterations or total loss of CFTR protein function, resulting in defectsin osmolarity, pH and redox properties of exocrine secretions. In thelungs, CF manifests itself by the presence of a thick mucus secretionwhich clogs the airways. In other exocrine organs, such as the sweatglands, CF may not manifest itself by an obstructive phenotype, butrather by abnormal salt composition of the secretions (hence theclinical sweat osmolarity test to detect CF patients). The predominantcause of illness and death in cystic fibrosis patients is progressivelung disease. The thickness of CF mucus, which blocks the airwaypassages, is believed to stem from abnormalities in osmolarity ofsecretions, as well as from the presence of massive amounts of DNA,actin, proteases and prooxidative enzymes originating from a subset ofinflammatory cells, called neutrophils. Indeed, CF lung disease ischaracterized by early, hyperactive neutrophil-mediated inflammatoryreactions to both viral and bacterial pathogens. The hyperinflammatorysyndrome of CF lungs has several underpinnings, among which an imbalancebetween pro-inflammatory chemokines, chiefly IL-8, and anti-inflammatorycytokines, chiefly IL-10, has been reported to play a major role. SeeChmiel et al., Clin Rev Allergy Immunol. 3(1):5-27 (2002). Studies havereported that levels of TNF-α, IL-6 and IL-1β were higher in thebronchoalveolar lavage fluid of cystic fibrosis patients, than inhealthy control bronchoalveolar lavage fluid (Bondfield, T. L., et al.Am. J. Resp. Crit. Care Med. 152(1):2111-2118, 1995).

Injection fibrosis (IF) is a complication of intramuscular injectionoften occurring in the quadriceps, triceps and gluteal muscles ofinfants and children in which subjects are unable to fully flex theaffected muscle. It typically is painless, but progressive. Studies havereported that the glycoprotein osteopontin (OPN) plays a role in tissueremodeling (Liaw, L., et al. J. Clin. Invest, 101(7):1469-1478, 1998)and that this proinflammatory mediator induces IL-1β up-regulation inhuman monocytes and an accompanying enhanced production of TNF-α andIL-6 (Naldini, A., et al. J. Immunol. 177:4267-4270, 2006; Weber, G. F.,and Cantor, H. Cytokine Growth Factor Reviews. 7(3):241-248, 1996).

Endomyocardial disease (hyperosinophilic syndrome (HS)) is a diseaseprocess characterized by a persistently elevated eosinophil count (1500eosinophils/mm³) in the blood. HS simultaneously affects many organs.Studies have reported that IL-1β, IL-6 and TNF-α are expressed at highlevels in viral-induced myocarditis patients (Satoh, M., et al. VirchowsArchiv. 427(5):503-509, 1996). Symptoms may include cardiomyopathy, skinlesions, thromboembolic disease, pulmonary disease, neuropathy,hepatosplenomegaly (coincident enlargement of the liver and spleen), andreduced ventricular size. Treatment may include utilizingcorticosteroids to reduce eosinophil levels.

Mediastinal fibrosis (MF) is characterized by invasive, calcifiedfibrosis centered on lymph nodes that blocks major vessels and airways.MF is a late complication of histoplasmosis. Studies in murine models offibrosis have reported that IL-10 and TNF-α are elevated significantly(Ebrahimi, B, et al. Am. J. Pathol. 158:2117-2125, 2001).

Myelofibrosis (myeloid metaplasia, chronic idiopathic myelofibrosis,primary myelofibrosis) is a disorder of the bone marrow in which themarrow undergoes fibrosis. Myelofibrosis leads to progressive bonemarrow failure. The mean survival is five years and causes of deathinclude infection, bleeding, organ failure, portal hypertension, andleukemic transformation. It has been reported that TNF-α and IL-6 levelsare elevated in animal models of viral-induced myelofibrosis(Bousse-Kerdiles, M., et al. Ann. Hematol. 78:434-444, 1999).

Retroperitoneal fibrosis (Ormond's disease) is a disease featuring theproliferation of fibrous tissue in the retroperitoneum. Theretroperitoneum is the body compartment containing the kidneys, aorta,renal tract, and other structures. It has been reported that IL-1, IL-6and TNF-α have key roles in the pathogenesis of retroperitoneal fibrosis(Demko, T., et al, J. Am. Soc. Nephrol. 8:684-688, 1997). Symptoms ofretroperitoneal fibrosis may include, but are not limited to, lower backpain, renal failure, hypertension, and deep vein thrombosis.

Nephrogenic systemic fibrosis (NSF, nephrogenic fibrosing dermopathy)involves fibrosis of the skin, joints, eyes and internal organs. NSF maybe associated with exposure to gadolinium. Patients develop large areasof hardened skin with fibrotic nodules and plaques. Flexion contractureswith an accompanying limitation of range of motion also may occur. NSFshows a proliferation of dermal fibroblasts and dendritic cells,thickened collagen bundles, increased elastic fibers, and deposits ofmucin. Some reports have suggested that a proinflammatory state providesa predisposing factor for causing nephrogenic systemic fibrosis (Saxena,S., et al. Int. Urol. Nephrol. 40:715-724, 2008), and that the level ofTNF-α is elevated in animal models of nephrogenic systemic fibrosis(Steger-Hartmann, T., et al. Exper. Tox. Pathol. 61(6): 537-552, 2009).

4. Risk Factors

4.1. Primary Risk Factors

4.1.1. Cigarette Smoking

While a number of risk factors for fibrotic airway diseases have beenidentified (some of which may play a role in their causation), tobaccosmoke remains the principal and most important cause of COPD. Thegreater the number of cigarettes smoked, the greater is the risk ofdeveloping fibrotic airwary diseases. An overwhelming majority of peoplewho develop fibrotic airway diseases are smokers, and their lungfunction decreases faster than that of non-smokers.

The most effective intervention is to stop smoking, preferably at anearly stage. Smokers who quit will not recover lost lung function, butthe rate of decline may revert to that of a non-smoker. Stopping smokingat an early stage improves the prognosis, regardless of how manyattempts are needed to quit. Individual susceptibility to developingfibrotic airwary diseases. in relation to cigarette smoking varies.Approximately 15% of smokers will develop clinically significant COPD,while approximately 50% will never develop any symptoms. The decrease inlung function is gradual, and the disease is usually diagnosed latebecause patients may adapt to symptoms of shortness of breath, or maynot notice the symptoms. Studies have shown that depending on the numberof cigarettes smoked per day, 24-47% of smokers develop airflowobstruction. Exposure to passive smoking increases susceptibility to thedisease.

4.1.2. Alpha-1 Antitrypsin Deficiency

This rare inherited condition results in the complete absence of one ofthe key antiprotease protection systems in the lung. It is a recessivedisorder affecting 1:4000 of the population. Patients with alpha-1antitrypsin deficiency are at risk of developing emphysema at an earlyage-between the ages of 20 and 40 years- and often have a strong familyhistory of the disease. Patients with the deficiency and emphysemainherit one abnormal gene from each parent; that is to say, the parentsare carriers of the gene. Such parents will have half the normal levelsof the antitrypsin in the blood, which may be enough to protect fromdeveloping emphysema. Likewise, all the children of an alpha-1antitrypsin deficient patient will carry one abnormal gene, but will notbe affected. The two common forms of alpha-1 antitrypsin deficiencyresult from point mutations in the gene that codes for alpha-1antitrypsin.

4.2. Associated Risk Factors

4.2.1. Environmental Pollution

There is strong evidence that fibrotic airwary diseases may beaggravated by air pollution, but the role of pollution in the etiologyof fibrotic airwary diseases is small when compared with that ofcigarette smoking. Air pollution with heavy particulate matter, carbon,and sulphur dioxide, which are produced by the burning of coal andpetroleum fossil fuels, are important causes or cofactors in thedevelopment of fibrotic airwary diseases. These originate mainly fromvehicle exhaust emissions, and photochemical pollutants such as ozone,in particular, are to be blamed. Indoor air pollution from biomass fuelburned for cooking and heating in poorly ventilated homes may be animportant risk factor for fibrotic airwary diseases, such as COPD, indeveloping countries, in particular for women.

4.2.2. Occupational Factors

Some occupations in which workers are exposed to coal, silica andcation, such as miners, textile workers and cement workers, areassociated with an increased risk of fibrotic airwary diseases. Exposureto cadmium, a heavy metal, and welding fumes has been recognized as acause of emphysema since the 1950s.

Many dusty occupations are more hazardous than exposure to gas or fumesand are associated with the development of chronic bronchitis andvarious forms of airway obstructive disease. Shipyard welders andcaulkers are also known to have an increased risk of developing fibroticairwary diseases, as well as those working in the constructionindustries that are exposed to cement dust.

4.2.3. Childhood Respiratory Infections

Chest infections in the first year of life, such as pneumonia andbronchiolitis, may predispose to the development of COPD in later life.This may be as a result or incomplete development of the respiratorysystem at birth until lung growth ends in early adulthood. If developinglungs are damaged, maximum potential lung function will not be achieved,producing symptoms of COPD at an early age.

4.3. Other Risk Factors

Other risk factors, which may play a role in causation and/or serves asearly symptoms of fibrotic airway diseases, such as pulmonary fibroses,include hypersensitivity pneumonitis (most often resulting from inhalingdust contaminated with bacterial, fungal, or animal products), sometypical connective tissue diseases (such as rheumatoid arthritis,systemic lupus erythematosus (SLE) and scleroderma), other diseases thatinvolve connective tissue (such as sarcoidosis and Wegener'sgranulomatosis), infections, certain medications (e.g. amiodarone,bleomycin, busulfan, methotrexate, and nitrofurantoin), and radiationtherapy to the chest.

5. Liver Tissue Remodeling

Liver tissue remodeling is of clinical interest because chronic damageto the liver induces necrosis or the parenchyma with consequentincreased deposition of extracellular matrix (ECM) that underlies theoccurrence of liver cirrhosis, hepatocellular carcinoma and liverfibrosis (Moshage H., J. Pathol. 1997; 181: 257-266). During remodeling,the liver undergoes rearrangement of the original architecture, withconsequent changes in the spatial organization and redefinition of thehistological borders (Giannelli G. et al., Histol. Histopathol. 2003;18: 1267-1274). The break-down of tissue boundaries and rearrangement oftissue architecture seem to be regulated by matrix metalloproteinases(MMPs), a large family of zinc-endopeptidases with proteolytic activitytoward ECM components (Giannelli G. et al., Histol. Histopathol. 2003;18: 1267-1274).

One member of the MMP family, MMP2, is found throughout the human bodyand possesses a wide spectrum of action toward the ECM (Giannelli G. etal., Histol. Histopathol. 2003; 18: 1267-1274). It is secreted as apro-enzyme and activated at the cellular surface by a membrane-type MMP(MT1-MMP) with the aid of an MMP inhibitor, tissue inhibitor ofmetalloproteinase-2 (TIMP2) (Strongin A. Y., et al., Biol. Chem. 1993;268: 14033-14039). The proteolytic activity of MMP2 is balanced by thepresence of TIMP2, which not only activates MMP2 by facilitating bindingof MMP2 to MT1-MMP but also inhibits MMP-2, creating a feedback systemin the regulation of ECM proteolysis (Fridman R. et al., Biochem. J.1993; 289(Pt 2): 411-416).

Inflammation and mediators of inflammation, also play a role in livertissue remodeling (Giannelli G. et al., Histol. Histopathol. 2003; 18:1267-1274). It has been reported that a number of different cytokines,including tumor necrosis factor (TNF)-α, interleukin (IL)-1β,transforming growth factor (TGF)-β1 and platelet-derived growth factor(PDGF) are up-regulated in liver parenchyma during chronic hepatitis(Castilla A. et al., N. Engl. J. Med. 1991; 324: 933-940). The alterednetwork of cytokines is thought to play a role not only in mediatinginflammation, but also in modulating the expression of MMPs and TIMPs,thereby influencing the local proteolysis of ECM proteins (Knittel T. etal., J. Hepatol. 1999; 30: 48-60).

5.1 Liver Fibrosis

The pathogenesis of hepatic fibrosis involves significant deposition offibrillar collagen and other extracellular matrix proteins. It is adynamic process of wound healing in response to a variety of persistentliver injuries caused by factors such as ethanol intake, viralinfection, drugs, toxins, cholestasis and metabolic disorders. Liverfibrosis distorts the hepatic architecture, decreases the number ofendothelial cell fenestrations and causes portal hypertension (MormoneE. et al., Chem. Biol. Interact. 2011 Sep. 30; 193(3): 225-231). Keyevents are the activation and transformation of quiescent hepaticstellate cells into myofibroblast-like cells with the subsequentup-regulation of proteins such as α-smooth muscle actin, interstitialcollagens, matrix metalloproteinases, tissue inhibitor ofmetalloproteinases and proteoglycans (Mormone E. et al., Chem. Biol.Interact. 2011 Sep. 30; 193(3): 225-231). Oxidative stress is a majorcontributing factor to the onset of liver fibrosis, and it is typicallyassociated with a decrease in the antioxidant defense (Mormone E. etal., Chem. Biol. Interact. 2011 Sep. 30; 193(3): 225-231). Currently,there is no effective therapy for advanced liver fibrosis. In its earlystages, liver fibrosis is reversible upon cessation of the causativeagent; recent developments in our understanding of the process ofhepatic fibrinogenesis suggest that a capacity for recovery fromadvanced fibrosis is possible (Pellicoro A, et al., Fibrogenesis &Tissue Repair 2012, 5(Suppl 1): S26; Benyon R C and Iredale J P, Gut2000; 46: 443-446 (April)).

5.1.1 Etiology

Most chronic liver diseases are associated with fibrosis and arecharacterized by parenchymal damage and inflammation. Alcohol abuse,chronic viral hepatitis (HBV and HCV), obesity, autoimmune hepatitis,parasitic diseases (e.g., schistosomiasis), metabolic disorders (e.g.,hemochromatosis and Wilson's disease), biliary disease, persistentexposure to toxins and chemicals and drug-induced chronic liver diseasesare the most common causes of hepatic fibrosis (Mormone E. et al., Chem.Biol. Interact. 2011 Sep. 30; 193(3): 225-231).

5.1.1.1 Alcohol Consumption

Alcohol consumption is a predominant etiological factor in thepathogenesis of chronic liver diseases worldwide, resulting in fattyliver, alcoholic hepatitis, fibrosis/cirrhosis, and hepatocellularcarcinoma (Miller A M, et al., Alcoholism-Clinical and ExperimentalResearch 2011; 35(5): 787-793). Acetaldehyde, the product of alcoholmetabolism, increases the secretion of transforming growth factor β1(TGFβ1) and induces TGFβ type II receptor expression in hepatic stellatecells (HSC), the key collagen-producing cell within the liver (Anania FA, et al., Arch. Biochem. Biophys. 1996; 331(2): 187-193). TGFβ1 is acritical factor in the progression of alcoholic liver disease (ALD) inpatients with steatosis and steatohepatitis and plays a significant roleas mediator of alcohol-induced liver fibrosis (Weng H L, et al.,Hepatology 2009; 50(1): 230-243). Acetaldehyde also selectively inducesphosphorylation of Smad3 but not of Smad2 (Greenwel P, et al.,Hepatology 2000; 31(1): 109-116). Both ethanol and acetaldehyde inducethe COL1A2 promoter and up-regulate collagen I protein expression (ChenA, Biochem. J. 2002; 368(Pt 3): 683-693).

Hepatic alcohol metabolism generates reactive oxygen species (ROS)causing significant cell death (Parola M and Robino G, J. Hepatol. 2001;35(2): 297-306). Oxidative stress promotes hepatocyte necrosis and/orapoptosis. Generation of ROS within hepatocytes may be a consequence ofan altered metabolic state, as it occurs in nonalcoholic fatty liverdisease and non-alcoholic steatohepatitis (Mormone E. et al., Chem.Biol. Interact. 2011 Sep. 30; 193(3): 225-231). Alternatively, it couldresult from ethanol metabolism as in alcoholic steatohepatitis, with ROSbeing generated mainly by the mitochondrial electron transport chain,cytochrome P450 isoforms such as cytochrome P4502E1 (CYP2E1), damagedmitochondria, xanthine oxidase, NADPH oxidase and generation of lipidperoxidation-end products (Haorah J, et al., Free Radic. Biol. Med.2008; 45(11): 1542-1550). In addition, it is known that chronic alcoholconsumption lowers glutathione levels, thus contributing to liver injury(Cederbaum A I, World J. Gastroenterol. 2010; 16(11): 1366-1376).ROS-derived mediators released by damaged neighboring cells can directlyaffect hepatic stellate cell (HSC) behavior. ROS up-regulate theexpression of critical genes related to fibrogenesis, includingpro-collagen type I, monocyte chemoattractant protein 1 (MCP-1) andtissue inhibitor of metalloproteinase-1 (TIMP1), possibly via activationof a number of critical signal transduction pathways and transcriptionfactors, including c-jun N-terminal kinases (JNKs), activator protein 1(AP-1) and nuclear factor kappa B (NFκB) (Bataller R, et al., Journal ofClinical Investigation 2005; 115(2): 209-218).

5.1.1.2 Chronic Viral Hepatitis

Chronic hepatitis B virus (HBV) and C virus (HCV) are the most commoncauses of liver disease worldwide, with an estimated 350 and 170 millionindividuals, respectively, with chronic infection (Custer B, et al., J.Clin. Gastroenterol. 2004; 38(10): S158-S168). In both cases, there issignificant chronic liver injury with subsequent progression to advancedliver fibrosis.

5.1.1.3 Other Causes of Liver Fibrosis

Other factors contributing to hepatic fibrosis are obesity andsteatosis, which can lead to nonalcoholic fatty liver disease and tochronic steatohepatitis. Nonalcoholic fatty liver disease has also beenreported in non-obese individuals in developing countries (Das K, etal., Hepatology, 2010; 51(5): 1593-1602).

Autoimmune hepatitis, which is the result of anomalous presentation ofhuman leukocyte antigen (HLA) class II in hepatocytes, causescell-mediated immune responses against the host liver, which can lead toliver fibrosis (Lim Y S, et al., J. Hepatology, 2008; 48(1): 133-139).Parasitic infections, like schistosomiasis, also have been shown totrigger advanced liver fibrosis and portal hypertension (Andersson K Land Chung R T, Curr Treat. Options Gastroenterol., 2007; 10(6):504-512).

Metabolic disorders, such as hemochromatosis and Wilson's disease, aretypically accompanied by chronic hepatitis and fibrosis (Lefkowitch J H,Curr. Opin. Gastroenterol., 2006; 22(3): 198-208). In hereditaryhemochromatosis, the excessive absorption and accumulation of iron intissues and organs, including liver, is related to mutations in the HFE(High-iron) gene (Feder J N, et al., J. Hepatol., 2003; 38(6): 704-709).Pathological accumulation of iron exacerbates oxidative stress resultingin increased lipid peroxidation. This leads to destruction of organellemembranes and in turn, cell death via hepatocyte necrosis and/orapoptosis (Sidorska K, et al., J. Biotech., Computational Bio. AndBionanotech., 2011; 92(1): 54-65). Wilson's disease or hepatolenticulardegeneration is a genetic disorder leading to copper accumulation in theliver and is due to a mutation in the APTase (ATP7B) that transportscopper (Zhang S, et al., Hum. Mol. Genet., 2011 Aug. 15; 20(16):3176-3187). Wilson's disease patients are characterized by liverfibrosis caused by severe mitochondrial changes, associated with anincreased number of peroxisomes, cytoplasmic lipid droplets and thepresence of lipolysosomes, characteristic cytoplasmic bodies formed bylipid vacuoles surrounded by electron-dense lysosomes. Nuclei arefrequently involved, with disorganization of the nucleoplasm and withglycogen inclusions (Fanni D, et al., Curr. Med. Chem., 2014; DOI:10.2174/0929867321666140601163244).

Cholestasis due to bile duct obstruction leads to chronic portalfibrosis and eventually cirrhosis. Moreover, chronic exposure to toxinsor chemicals such as N-nitrosodimethylamine, carbon tetrachloride (CCl₄)or thioacetamide leads to severe hepatic fibrosis in experimental animalmodels (George J, et al., Gene Therapy, 2007; 14(10): 790-803;Domoenicali M, et al., J. Hepatol., 2009; 51(6): 991-999; Salguero P R,et al., Lab. Invest. 2008; 88(11): 1192-1203). Exposure to thesechemicals in humans is rare and generally occurs in industries wherethese chemicals are routinely used (Mormone E. et al., Chem. Biol.Interact. 2011 Sep. 30; 193(3): 225-231).

5.2. Cell Types Involved in the Pathogenesis of Liver Fibrosis

5.2.1 Hepatic Stellate Cells

Hepatic stellate cells (HSCs) reside in the space of Disse betweenhepatocytes and sinusoidal endothelial cells (Friedman S L,Gastroenterology, 2008; 134(6): 1655-1669). Quiescent HSCs arecharacterized by significant expression of desmin and vitamin A storage.Following liver injury, HSCs lose their vitamin A content, increaseexpression of α-smooth muscle actin (α-SMA), acquire amyofibroblast-like phenotype, become proliferative, motile,pro-fibrogenic, and contractile, and show abundant rough endoplasmicreticulum (Gressner A M, Kidney International, 1996; 49: S39-S45).

Many factors are known to contribute to activation of HSCs. Damage tohepatocytes and Kupffer cell activation are still considered the primaryeffectors driving HSC activation (Nieto N, et al., J. Biol. Chem. 2002;277(12): 9853-9864; Nieto N, Hepatology, 2006; 44(6): 1487-1501).Mediators released from damaged hepatocytes, such as lipid peroxidationproducts, intermediate metabolites of drugs or hepatotoxins,acetaldehyde and 1-hydroxyethyl radical from alcohol metabolism, as wellas ROS (e.g., hydrogen peroxide and superoxide radicals) are stronginducers of HSC activation (Mormone E. et al., Chem. Biol. Interact.2011 Sep. 30; 193(3): 225-231).

Activated Kupffer cells release ROS and cytokines that are crucial forHSC activation as well (Nieto N, Hepatology, 2006; 44(6): 1487-1501).They are a major source of TGFβ and platelet-derived growth factor(PDGF), two potent profibrogenic cytokines that traditionally have beenconsidered key fibrogenic and proliferative stimuli to HSC (Tsukamoto H,Alcoholism-Clinical and Experimental Research, 1992; 23(5): 911-916).Kupffer cell phagocytic activity generates large amounts of ROS thatcould further activate HSC and induce their fibrogenic potential.Furthermore, it has been shown that the addition of ethanol andarachidonic acid synergized to activate Kupffer cells modulated thefibrogenic response by a mechanism involving TNFα, reduced glutathione,and TGFβ (Cubero F J and Nieto N, Hepatology, 2008; 48(6): 2027-2039).

Likewise, cytochrome P450 2E1-dependent generation of ROS has been shownto be critical for increased collagen I protein synthesis in co-culturesof hepatocytes and HSCs (Nieto N, et al., J. Biol. Chem., 2002; 277(12):9853-9864).

5.2.2 Portal Fibroblasts

The portal connective tissue in healthy liver is surrounded by quiescentportal fibroblasts, which constitute a second population of liver cellsimplicated in portal fibrosis (Tuchweber B, et al., LaboratoryInvestigation, 1996; 74(1): 265-278). Derived from small portal vessels,they express markers distinct from HSC (e.g. elastin) (Li Z, et al.,Hepatology, 2007; 46(4): 1246-1256). Proliferation of biliary cells isoften accompanied by proliferation of portal fibroblasts, which formonion-like configurations around biliary structures and acquire amyofibroblast phenotype. These cells are thought to be involved in theearly deposition of extracellular matrix (ECM) in portal zones(Desmouliere A, et al., Lab Invest, 1997; 76(6): 765-778).

5.2.3 Bone Marrow-derived Mesenchymal Stem Cells

Evidence suggests that bone marrow-derived stem cells are recruitedduring both progression and regression of liver fibrosis. Duringregression from CCl₄-induced hepatic fibrosis, bone marrow-derivedmesenchymal stem cells migrate into the fibrotic liver, where they canexpress matrix metalloprotease-13 (MMP13) and MMP9 (Cheng Y J, et al.,Life Sci., 2009; 85(13-14): 517-525). In addition, granulocytecolony-stimulating factor (G-CSF) and hepatocyte growth factor (HGF)treatment significantly enhances migration of bone marrow-derived cellsinto the fibrotic liver and accelerates the regression of liver fibrosis(Higashiyama R, et al., Hepatology, 2007; 45(1): 213-222).Over-expression of hepatocyte growth factor (HGF) together withgranulocyte colony-stimulating factor (G-CSF), synergistically stimulateMMP9 expression, which is followed by accelerated resolution of fibroticscars (Bird T G, et al., Cell Tissue Res., 2008; 331(1): 283-300).Although a significant contribution of bone marrow-derived cells hasbeen shown in human liver fibrosis, it is unclear what type ofmesenchymal stem cells are involved (Forbes S J, et al.,Gastroenterology, 2004; 126(4): 955-963).

5.2.4 Hepatocytes and Biliary Epithelial Cells

Epithelial-to-mesenchymal transition (EMT) is emerging as a possiblesource of injury-associated mesenchymal cells derived either fromresident hepatocytes or from biliary epithelial cells (Dooley S, et al.,Gastroenterology, 2008; 135(2): 642-659; Nitta T, et al., Hepatology,2008; 48(3): 909-919). The main molecules inducing EMT are TGFβ,epidermal growth factor (EGF), insulin-like growth factor-II (IGF-II)and fibroblast growth factor-2 (FGF-2) (Zeisberg M, et al., J. Biol.Chem., 2007; 282(32): 23337-23347). Hepatocytes that express albuminalso express fibroblast-specific protein-1 (FSP 1) in response to CCl₄in vivo or to TGFβ1 in vitro (Zeisberg M, et al., J. Biol. Chem., 2007;282(32): 23337-23347). It has been reported that hepatocytes expressCOL1A1 in response to TGFβ1 in vitro, and that Smad signaling mediatesEMT (Kaimori A, et al., J. Biol. Chem., 2007; 282(30): 22089-22101).

Biliary epithelial cells have been described to be involved in EMT inliver fibrogenesis. In primary biliary cirrhosis, it has been shown thatcells of the bile duct express fibroblast-specific protein-1 (FSP 1) andvimentin, early markers of fibroblasts (Robertson H, et al., Hepatology,2007; 45(4): 977-981). A consequence of EMT in biliary epithelial cellsis the amplification of the pool of portal fibroblasts, contributingsignificantly to portal fibrosis. In vitro studies with human biliaryepithelial cells have confirmed these clinical observations (Rygiel K A,Lab. Invest., 2008; 88(2): 112-123). Thus, EMT could be considered amechanism participating in the pathogenesis of chronic cholestatic liverdisease.

5.2.5 Fibrocytes

Fibrocytes are a circulating, bone marrow-derived, CD34⁺ cellsubpopulation with fibroblast-like properties initially associated withtissue repair in subcutaneous wounds (Bucala R. et al., Mol. Med. 1994;1(1): 71-81). They comprise a fraction of about 1% of the circulatingpool of leukocytes expressing markers of mesenchymal cells (Moore B B,et al., Am. J. Pathol., 2005; 166(3): 675-684). It has been hypothesizedthat bone marrow-derived circulating CD34⁺ fibrocytes represent keymediators of liver fibrogenesis in the Abcb4^(−/−) mice, whichrepresents a reproducible, well-characterized non-surgical mouse modelfor cholangiopathy in humans (Roderfeld M, et al., Hepatology, 2010;51(1): 267-276).

5.3 Molecular Pathogenesis of Liver Fibrosis

5.3.1 Cell-Cell and Cell-Matrix Interactions

Alterations in normal cell-cell and cell-matrix interactions play asignificant role in pathogenesis of liver fibrosis. When normalcell-cell and cell-matrix interactions are altered due to hepatocytenecrosis or invasion of inflammatory cells, new interactions areestablished that trigger a fibrogenic response (Mormone E. et al., Chem.Biol. Interact. 2011 Sep. 30; 193(3): 225-231). In the fibrotic liver,significant quantitative and qualitative changes occur in thecomposition of ECM in the periportal and perisinusoidal areas (ZeisbergM, et al., Mol. Cell Biochem., 2006; 283(1-2): 181-189). Fibrotic scarsare typically composed of fibrillar collagen type I and III,proteoglycans, fibronectin and hyaluronic acid (George J, et al.,International J. Biochem. & Cell Biol., 2004; 36(2): 307-319). As aresult, alteration in the physiological architecture of the liveroccurs, particularly in the space of Disse, where the low electron-denseECM is replaced by one rich in fibrillar collagens and fibronectin. Thisleads to loss of endothelial cell fenestrations, impaired exchange ofsolutes among neighboring cells, altered hepatocyte function andsubsequent non-parenchymal cell damage (Hernandez-Gea V. and Friedman SL, Annu. Rev. Pathol. 2011; 6: 425-456).

5.3.2 Oxidative Stress

The term “oxidative stress” as used herein refers to a disturbance inthe balance between the production of reactive oxygen species (freeradicals) and antioxidant defenses.

Chronic HBV infection and long-term consumption of alcohol induce celldamage through increased generation of reactive oxygen species (ROS)(Muriel P, Hepatol. Int. 2009; 3(4): 526-536).

Oxidative stress, which favors mitochondrial permeability transition, isable to promote hepatocyte necrosis and/or apoptosis (Mormone E. et al.,Chem. Biol. Interact. 2011 Sep. 30; 193(3): 225-231). It has beenhypothesized that in some clinically relevant conditions, generation ofROS within hepatocytes may represent an altered metabolic state as innon-alcoholic fatty liver disease and non-alcoholic steatohepatitis, orsignificant ethanol metabolism as it occurs in alcoholic steatohepatitis(Mormone E. et al., Chem. Biol. Interact. 2011 Sep. 30; 193(3):225-231).

ROS are generated mainly via the mitochondrial electron transport chainor via activation of cytochrome P450 (mostly cytochrome P450 2E1), NADPHoxidase, xanthine oxidase or via mitochondrial damage. The ROS generatedcan directly affect HSC and myofibroblast behavior (Tilg H andHotamisligil G S, Gastroenterology, 2006; 131(3): 934-945; Nieto N,Hepatology, 2006; 44(6): 1487-1501). ROS up-regulate the expression ofcritical fibrosis-associated genes such as COL1A1, COL1A2, MCP1 andTIMP1 via activation of signal transduction pathways and transcriptionfactors, including JNK, activator protein-1 (AP-1) and NFκB (Bataller Rand Brenner D A, Journal of Clinical Investigation, 2005; 115(2):209-218). ROS generation in HSCs and myofibroblasts occurs in responseto several known pro-fibrogenic mediators, including angiotensin II,platelet-derived growth factor (PDGF), TGFβ and leptin (De Minicis S, etal., Gastroenterology, 2006; 131(1): 272-275). Overall, a decrease inthe antioxidant defense system, such as GSH, catalase or SOD, inconjunction with enhanced lipid peroxidation, leads to a pro-fibrogenicresponse by enhancing collagen I protein expression (George J, Clin.Chim. Acta, 2003; 335(1-2): 39-47).

p38 mitogen-activated protein kinases (MAPK) may be essential in theup-regulation of proinflammatory cytokines and can be activated bytransforming growth factor β (TGFβ), tumor necrosis factor-α (TNFα),interleukin-1β (IL-1β), and oxidative stress (Tormos A M, et al.,Hepatology, 2013; 57(5): 1950-1961). Substrates of p38 include proteinkinases, such as MAPKAP kinase 2 (MK2) and MK5, and transcriptionfactors, such as ATF2, p53, and Mitf (Hui L, et al., Cell Cycle, 2007;6(20): 2429-2433). These diverse targets mediate the activated p38signal to various types of cellular functions such as differentiation,apoptosis, cytokine production, and cell cycle control (Nakagawa H andMaeda S, Pathology Research International, 2012; doi:10.1155/2012/172894). Recent in vivo studies have shown thatstress-activated mitogen-activated protein kinase (MAPK) signalingconverging on c-Jun NH2-terminal kinase (JNK) and p38 plays a centralrole in inflammation-mediated liver injury and compensatory hepatocyteproliferation (Sakurai T, et al., PNAS, 2006; 103(28): 10544-10551; HuiL, et al., Nature Genetics, 2007; 39(6): 741-749; Hui L, et al., JCI,2008; 118(12): 3943-3953; Sakurai T, et al., Cancer Cell, 2008; 14(2):156-165; Maeda S, Gastroenterology Research and Practice, 2010; doi:10.1155/2010/367694; Wagner E F and Nebreda A R, Nature Reviews Cancer,2009; 9(8): 537-549; Min L, et al., Seminars in Cancer Biology, 2011;21(1): 10-20).

5.3.3 MMPs and TIMPs

The ECM is a highly dynamic milieu subject to constant remodeling.Life-threatening pathological conditions arise when ECM remodelingbecomes excessive or uncontrolled. Among the cells involved in hepaticECM degradation are HSCs, neutrophils and macrophages. MMPs are the mainenzymes responsible for ECM degradation (Goto T, et al.,Pathophysiology, 2004; 11(3): 153-158). Tissue inhibitors of matrixmetalloproteinase-1 (TIMPs) have the ability to inhibit MMPs (Goto T, etal., Pathophysiology, 2004; 11(3): 153-158). Therefore, regulation ofthe MMP-TIMP balance is crucial for efficient ECM remodeling. TheMMP-TIMP balance becomes tipped in response to multiple pro-fibrogenicinsults. Activated HSCs not only synthesize and secrete ECM proteinssuch as collagens type I and type III, but also produce MMP1 and MMP13(Iredale J P, et al., Clin. Sci. (Lond), 1995; 89(1): 75-81; Knittel T,et al., Hisotchem. Cell Biol., 2000; 113(6): 443-453). However, MMP1 andMMP13 expression decreases as HSC activation progresses, while theactivity of other MMPs remains relatively constant, except for MMP2 andMMP9 (Benyon R C and Arthur M J, Semin. Liver Dis. 2001; 21(3):373-384). The increase in MMP2 activity is associated with distortion ofthe normal lobular architecture, which further activates HSC (Benyon R Cand Arthur M J, Semin. Liver Dis. 2001; 21(3): 373-384). Activated HSCsup-regulate the expression and synthesis of TIMP1 and TIMP2 (Iredale JP, et al., Hepatology, 1996; 24(1): 176-184). TIMP1 not only preventsthe degradation of the rapidly increasing ECM by blocking MMPs, but alsoinhibits the apoptosis of activated HSCs (Murphy F R, et al., J. Biol.Chem., 2002; 277(13): 11069-11076). The net result is the deposition ofmature collagen fibers within the space of Disse which results inscarring.

6. Renal Fibrosis

Renal fibrosis is the result of excessive accumulation of extracellularmatrix that occurs in nearly every type of chronic kidney disease (LiuY, Kidney International 2006; 69: 213-217). The progression from chronickidney disease to renal fibrosis often results in widespread tissuescarring, complete destruction of kidney parenchyma, and end-stage renalfailure, a condition that requires dialysis or kidney replacement(Schieppati A et al., Kidney International 2005; 68(Suppl 1): S7-S10).Renal fibrosis represents a failed wound-healing process of kidneytissue after chronic, sustained injury. Several cellular pathways,including mesangial and fibroblast activation, as well as tubularepithelial-mesenchymal transition, have been identified as generatingthe matrix-producing cells in renal disease conditions (Liu Y, KidneyInternational 2006; 69: 213-217).

6.1 Cellular Events in Renal Fibrosis

Renal fibrosis is often pathologically described as glomerulosclerosis,tubule-interstitial fibrosis, inflammatory infiltration and loss ofrenal parenchyma characterized by tubular atrophy, capillary loss andpodocyte depletion (Liu Y, Kidney International 2006; 69: 213-217). Theunderlying cellular events leading to these histologic presentationsinclude mesangial and fibroblast activation, tubular epithelial tomesenchymal transition (EMT), monocyte/macrophage, and T-cellinfiltration, and cell apoptosis (Eddy A A, Pediatr. Nephrol. 2000; 15:290-301; Iwano M and Neilson E G, Curr Opin. Nephrol. Hypertens. 2004;13: 279-284; Hirschberg R, J. Am. Soc. Nephrol. 2005; 16: 9-11; Liu Y,J. Am. Soc. Nephrol. 2004; 15: 1-12).

6.2 Molecular Pathogenesis of Renal Fibrosis

Although more than a dozen different fibrogenic factors have beenimplicated in the pathogenesis of renal fibrosis, including variouscytokines and hormonal, metabolic, and hemodynamic factors, it is widelyaccepted that transforming growth factor-β (TGF-β) and its downstreamSmad signaling mediators play an essential role (Bottinger E P andBitzer M, J. Am. Soc. Nephrol. 2002; 13: 2600-2610; Schnaper H W et al.,Am. J. Physiol. Renal. Physiol. 2003; 284: F243-F252). In vitro, TGF-βas a sole factor can stimulate mesangial cells, interstitialfibroblasts, and tubular epithelial cells to undergo myofibroblasticactivation or transition, to become matrix-producing fibrogenic cells(Liu Y, Kidney International 2006; 69: 213-217; Bottinger E P and BitzerM, J. Am. Soc. Nephrol. 2002; 13: 2600-2610; Schnaper H W et al., Am. J.Physiol. Renal. Physiol. 2003; 284: F243-F252). Expression of exogenousTGF-β, either via gene delivery in vivo or in transgenic mice, causesrenal fibrosis (Liu Y, Kidney International 2006). Conversely,inhibition of TGF-β by multiple strategies suppresses renal fibroticlesions and prevents progressive loss of kidney function (Liu Y, KidneyInternational 2006; 69: 213-217; Bottinger E P and Bitzer M, J. Am. Soc.Nephrol. 2002; 13: 2600-2610; Schnaper H W et al., Am. J. Physiol.Renal. Physiol. 2003; 284: F243-F252). TGF-β induction also appears tobe a convergent pathway that integrates, directly or indirectly, theeffects of many other fibrogenic factors. Some of these, such asangiotensin II and high glucose, act as an upstream TGF-β inducer,whereas others, such as connective tissue growth factor, work as itsdownstream effector (Liu Y, Kidney International 2006; 69: 213-217;Bottinger E P and Bitzer M, J. Am. Soc. Nephrol. 2002; 13: 2600-2610;Schnaper H W et al., Am. J. Physiol. Renal. Physiol. 2003; 284:F243-F252).

The TGF-β signal is transduced through its cell membrane type I and typeII serine/threonine kinase receptors (Bottinger E P and Bitzer M, J. Am.Soc. Nephrol. 2002; 13: 2600-2610). Receptor activation triggers thephosphorylation and activation of its downstream signaling mediators,Smad2 and Smad3 (Liu Y, Kidney International 2006). PhosphorylatedSmad2/3 bind to common partner Smad4, and subsequently translocate intonuclei, where they control the transcription of TGF-β-responsive genes(Liu Y, Kidney International 2006; 69: 213-217; Bottinger E P and BitzerM, J. Am. Soc. Nephrol. 2002; 13: 2600-2610; Schnaper H W et al., Am. J.Physiol. Renal. Physiol. 2003; 284: F243-F252). TGF-β/Smad signaling isregulated at both prereceptor and postreceptor stages through multiplelevels of modulation, which include TGF-β gene expression, latent TGF-βactivation, its receptor expression, and postreceptor Smad signaling(Liu Y, Kidney International 2006). In the fibrotic kidney, a multitudeof mechanisms lead to a hyperactive TGF-β/Smad signaling, includinginduction of TGF-β expression, enhanced post-translational activation ofTGF-β protein and its release from latent complexes. The receptors forTGF-β are also induced in diseased kidney (Liu Y, Kidney International2006; 69: 213-217).

Smad signaling in normal kidney is tightly constrained by a family ofproteins known as Smad transcriptional corepressors, which include SnoN,Ski, and TGIF (Liu Y, Kidney International 2006; 69: 213-217). Throughvarious mechanisms, these Smad antagonists effectively confineSmad-mediated gene transcription, thereby safeguarding the tissue fromunwanted TGF-β response (Liu Y, Kidney International 2006; 69: 213-217).Recently, it has been demonstrated that SnoN and Ski are progressivelydiminished in the fibrotic kidney, suggesting that the loss of Smadantagonists is an important mechanism that amplifies the TGF-β signal(Yang J et al., J. Am. Soc. Nephrol. 2003; 14: 3167-3177).

6.3 Matrix-Degrading Enzymes in Renal Fibrosis

It is generally believed that the excessive matrix accumulation seen inthe fibrotic kidney results from both overproduction of matrixcomponents and defects in its degradation. This notion is supported bymany observations that plasminogen activator inhibitor-1 and tissueinhibitor of matrix metalloproteinase-1 are often upregulated in thediseased kidney. Renal tissue produces a number of proteases, in whichthe plasminogen/plasmin and matrix metalloproteinase (MMP) systemsconstitute a proteolytic network that is capable of degrading allcomponents of matrix proteins (Liu Y, Kidney International 2006).

Given their proteolytic ability, matrix-degrading enzymes arehistorically considered to reduce matrix accumulation, therebyattenuating renal fibrosis after injury. However, recent genetic studiesusing knockout mice have painted a different and complex picture of thefunction of these proteins in relation to fibrotic lesions in vivo (LiuY, Kidney International 2006). It has been reported that ablation oftissue-type plasminogen activator (tPA) protects the kidney fromdeveloping interstitial fibrosis in obstructive nephropathy, which seemsto have little to do with its proteolytic activity (Yang J et al., J.Clin. Invest. 2002; 110: 1525-1538). The pathogenic effect of tPA inobstructive nephropathy primarily depends on its ability to induce MMP-9gene expression (Liu Y, Kidney International 2006). Increased MMP-9disrupts the integrity of tubular basement membrane, which leads to thepromotion of tubular EMT. Further investigations reveal that tPA is ableto bind to the cell membrane receptor low-density lipoproteinreceptor-related protein-1, induces its phosphorylation on tyrosineresidues, triggers intracellular signal transduction, andtrans-activates MMP-9 gene expression in renal interstitial fibroblasts(Liu Y, Kidney International 2006).

Plasmin, a serine protease that can directly degrade matrix proteins andactivate MMPs, is also thought to be beneficial in reducing renalfibrosis (Liu Y, Kidney International 2006). However, knockout of theplasminogen gene does not aggravate the fibrotic lesions after ureteralobstruction. Instead, mice lacking plasmin display a reduced collagenaccumulation, suggesting a significant pathogenic effect of this enzyme(Edgtton K L et al., Kidney International 2004; 66: 68-76).

MMP-2 also is found to be necessary and sufficient to induce tubular EMTin vitro (Cheng S and Lovett D H, Am. J. Pathol. 2003; 162: 1937-1949).Transgenic mice with overexpression of MMP-2 display fibrotic lesions(Liu Y, Kidney International 2006). A recent study also demonstratesthat MMP-3 induces Rac1 expression, which causes an increase in cellularreactive oxygen species and promotes EMT (Radisky D C et al., Nature2005; 436: 123-127).

7. Drug-Induced Kidney Injury

Various in vitro and in vivo studies have shown that the administrationof certain drugs acts as a stimulus to trigger various MAPK cascades,which in turn, mediate cellular responses to kidney injury (Naughton C.A., American Family Physician, vol. 78, no. 6, pp. 743-750, 2008).Activation and dysregulation of normal MAPK signaling pathways has beenimplicated in both acute and chronic kidney injury (Cassidy H et al.,Journal of Signal Transduction, vol. 2012, Article ID 463617, 15 pages).Renal biopsies in humans have shown upregulation of MAPKs in a varietyof renal conditions, suggesting involvement in human renal disease(Cassidy H et al., Journal of Signal Transduction, vol. 2012, Article ID463617, 15 pages).

7.1 Antibiotic-induced Kidney Injury

Acute kidney injury (AKI) is a common side effect of antibiotic therapy.Several studies have implicated the MAPK signaling cascade inantibiotic-induced renal injury initiated by several distinct classes ofantibiotics.

For example, Volpini et al. showed that MAPKs may be involved in thepathogenesis of acute renal failure following treatment with gentamicin(Volpini R. A. et al., Brazilian Journal of Medical and BiologicalResearch, vol. 39, no. 6, pp. 817-823, 2006). Briefly, the expression ofp-p38 MAPK and NF-κB in the kidney during the evolution oftubulointerstitial nephritis and its relationship with histologicalfeatures and renal function was investigated in gentamicin-treated ratsin the presence or absence of an NF-κB inhibitor. Data obtained in thisstudy showed that p38 MAPK expression is increased during thedevelopment of gentamicin-induced interstitial nephritis and that suchalteration is associated with enhancement of NF-κB expression and theinflammatory process in the renal cortex, suggesting the involvement ofthe p38 MAPK pathway in gentamicin-induced renal lesions (Volpini R. A.et al., Brazilian Journal of Medical and Biological Research, vol. 39,no. 6, pp. 817-823, 2006). Likewise, Ozbek et al. found that p38-MAPK isupregulated in rat kidneys following gentamicin treatment, and it hasbeen shown that combination treatment with the lipid-lowering drug,atorvastatin, ameliorated gentamicin-induced nephrotoxicity, throughinhibition of p38-MAPK and NF-κB expression (Ozbek E. et al., RenalFailure, vol. 31, no. 5, pp. 382-392, 2009).

A study investigating the effects of vancomycin exposure in renal LLCPK₁cells on cell proliferation showed a dose- and time-dependent increasein cell number and total cellular protein (Cassidy H et al., Journal ofSignal Transduction, vol. 2012, Article ID 463617, 15 pages; King D. W.and Smith M. A., Toxicology in Vitro, vol. 18, no. 6, pp. 797-803,2004). These effects were inhibited by pretreatment with the MAPKinhibitor, PD098059, thus preventing vancomycin-induced entry into thecell cycle. This data suggests an association between the cellproliferative effects of vancomycin and the induction of MAPK signalingcascades (Cassidy H et al., Journal of Signal Transduction, vol. 2012,Article ID 463617, 15 pages; King D. W. and Smith M. A., Toxicology inVitro, vol. 18, no. 6, pp. 797-803, 2004).

7.2 Calcineurin-Inhibitor-Induced Kidney Injury

The calcineurin inhibitors (CNIs) CsA and FK506 are widely used intransplant organ recipients. However, it has been shown that in kidneyallografts, CsA and FK506 cause tubulointerstitial, as well asmesangial, fibrosis (Sutherland B. W. et al., Oncogene, vol. 24, no. 26,pp. 4281-4292, 2005). The fibrogenic effect of CNIs in the renalallograft is predominantly mediated by elevated intrarenal expression ofTGF-β and subsequent excessive extracellular matrix (ECM) generation(Shihab F. S. et al., American Journal of Kidney Diseases, vol. 30, no.1, pp. 71-81, 1997; Ignotz R. A. and Massague J., Journal of BiologicalChemistry, vol. 261, no. 9, pp. 4337-4345, 1986; Schnaper H. W. et al.,American Journal of Physiology, vol. 284, no. 2, pp. F243-F252, 2003).Studies using rat kidney mesangial cells have shown that CsA and FK-506induce an extremely rapid and dose-dependent increase of Y-Box-bindingprotein-1 (YB-1) content in a cell type-specific manner. YB-1, a memberof the family of cold-shock proteins with mitogenic properties whichcontrols, among other things, TGF-β1 translation in proximal tubularcells, is a downstream target of MAPK ERK1/2 (Lu Z. H. et al., Molecularand Cellular Biology, vol. 25, no. 11, pp. 4625-4637, 2005; Fraser D. J.et al., Kidney International, vol. 73, no. 6, pp. 724-732, 2008; HanssenL. et al., Journal of Immunology, vol. 187, no. 1, pp. 298-308, 2011).

7.3 Chemotherapeutic Agent-induced Kidney Injury

Cancer chemotherapeutics include, without limitation, alkylating agents(e.g., cyclophosphamide); antimetabolites (e.g., methotrexate); plantalkaloids (e.g., etoposide); anthracyclines (e.g., doxirubicin);antitumor antibiotics (e.g., mitomycin C); platinum compounds (e.g.,cisplatin); and taxanes (e.g., taxol) (Chu E. and Devita Jr. V. T.,Eds., Physicians' Cancer Chemotherapy Drug Manual 2001, Jones andBartlett Publishers, London, U K, 2001). Chemotherapeutic agents cancause nephrotoxicity in various ways, with some drugs exerting immediateeffects on renal function while others are known to have cumulativeeffects, causing renal injury after long periods of use (Vogelzang N.J., Oncology, vol. 5, no. 10, pp. 97-105, 1991). For example, cisplatin,one of the most successful antineoplastic agents to date, causes acutekidney injury with clinically measureable nephrotoxicity usuallydetected 10 days after administration (Cassidy H et al., Journal ofSignal Transduction, vol. 2012, Article ID 463617, 15 pages).

It is believed that MAPK plays a pivotal role in cisplatin-inducednephrotoxicity. Arany et al. showed that ERK, and not p38 or JNK/SAPKinhibition, prevented cisplatin induced toxicity (Arany I. et al.American Journal of Physiology, vol. 287, no. 3, pp. F543-F549, 2004).Other studies have shown that pharmacological inhibition of p38 both invitro and in vivo prevented toxicity (Ramesh G. and Reeves W. B.,American Journal of Physiology, vol. 289, no. 1, pp. F166-F174, 2005;Francescato H. D. C. et al., Life Sciences, vol. 84, no. 17-18, pp.590-597, 2009). It also has been shown that JNK/SAPK inhibition resultsin a significant reduction in cisplatin-induced nephrotoxicity in vivo(Francescato H. D. C. et al., Nephrology Dialysis Transplantation, vol.22, no. 8, pp. 2138-2148, 2007). Pabla et al. identified PKCδ as acritical regulator of cisplatin nephrotoxicity. The data showed thatduring cisplatin nephrotoxicity, Src interacted with, phosphorylated,and activated PKCδ in mouse kidney lysates. After activation, PKCδregulated MAPKs, but not p53, to induce renal cell apoptosis. Thus,inhibition of PKCδ, pharmacologically or genetically, attenuated kidneycell apoptosis and tissue damage, preserving renal function duringcisplatin treatment. Conversely, inhibition of PKCδ enhancedcisplatin-induced cell death in multiple cancer cell lines and,remarkably, enhanced the chemotherapeutic effects of cisplatin inseveral xenograft and syngeneic mouse tumour models while protectingkidneys from nephrotoxicity (Pabla N. et al., Journal of ClinicalInvestigation, vol. 121, no. 7, pp. 2709-2722, 2011; Cassidy H et al.,Journal of Signal Transduction, vol. 2012, Article ID 463617, 15 pages).

7.4 Nonsteroidal Anti-Inflammatory Drug (NSAID)-Induced Kidney Injury

Nonsteroidal anti-inflammatory drugs (NSAIDs) include carboxylic acids(e.g., aspirin); acetic acids (e.g., diclofenac); propionic acids (e.g.,ibuprofen and ketoprofen); and Cox-2 inhibitors (e.g., celecoxib).NSAIDs are known to be nephrotoxic with the spectrum of nephrotoxicityincluding acute tubular necrosis, acute tubulointerstitial nephritis,glomerulonephritis, renal papillary necrosis, chronic renal failure,salt and water retention, hypertension, and hyperkalaemia (Perazella M.A. and Tray K., American Journal of Medicine, vol. 111, no. 1, pp.64-67, 2001; Ejaz P. et al., Journal of Association of Physicians ofIndia, vol. 52, pp. 632-640, 2004; Schier R. W. and Henrich W. L.,Journal of the American Medical Association, vol. 251, no. 10, pp.1301-1302, 1984).

Hou et al. investigated the molecular basis of the renal injury inducedby NSAIDs by evaluating the expression of the stress marker, hemeoxygenase-1 (HO-1), in celecoxib-stimulated glomerular mesangial cells(Ho C. C. et al., Annals of the New York Academy of Sciences, vol. 1042,pp. 235-245, 2005). Treatment with celecoxib resulted in aconcentration- and time-dependent increase of HO-1 expression (Ho C. C.et al., Annals of the New York Academy of Sciences, vol. 1042, pp.235-245, 2005). Conversely treatment with N-acetylcysteine, a freeradical scavenger, strongly decreased HO-1 expression, suggesting theinvolvement of reactive oxygen species (ROS) (Ho C. C. et al., Annals ofthe New York Academy of Sciences, vol. 1042, pp. 235-245, 2005).Treatment with various MAPK inhibitors showed that only a specific JNKinhibitor attenuated celecoxib-induced HO-1 expression (Ho C. C. et al.,Annals of the New York Academy of Sciences, vol. 1042, pp. 235-245,2005). Kinase assays demonstrated increased phosphorylation andactivation of c-JNK following NSAID treatment (Ho C. C. et al., Annalsof the New York Academy of Sciences, vol. 1042, pp. 235-245, 2005).Treatment with a PI-3K specific inhibitor prevented the enhancement ofHO-1 expression, which correlated with inhibition of the phosphorylationof the PDK-1 downstream substrate Akt/protein kinase B (PKB) (Ho C. C.et al., Annals of the New York Academy of Sciences, vol. 1042, pp.235-245, 2005). The results of this study suggested thatcelecoxib-induced HO-1 expression in glomerular mesangial cells may bemediated by ROS via the JNK-PI-3K cascade (Ho C. C. et al., Annals ofthe New York Academy of Sciences, vol. 1042, pp. 235-245, 2005).

8. Vascular Fibrosis

Vascular fibrosis is characterized by reduced lumen diameter andarterial wall thickening, which is attributed to excessive deposition ofextracellular matrix (ECM). It involves proliferation of vascular smoothmuscle cell (VSMC), accumulation of ECM and inhibition of matrixdegradation. It is associated with the renin-angiotensin-aldosteronesystem (RAAS), oxidative stress, inflammatory factors, growth factorsand imbalance of endothelium-derived cytokine secretion. Specifically,Angiotensin II (Ang II) and aldosterone (the circulating effectorhormones of RAAS) have been implicated in the pathophysiology ofvascular fibrosis. Transforming growth factor-beta (TGF-beta) has beenshown to play a critical role in ECM accumulation and vascularremodeling via up-regulation of connective tissue growth factor (CTGF)and fibroblast growth factor among others. An imbalance between matrixmetalloproteinases (MMPs) and tissue inhibitors of metalloproteinases(TIMPs) results in collagen accumulation and adverse matrix remodeling.Aberrant expression or function of peroxisome proliferator-activatedreceptor gamma (PPAR gamma) has also been reported to contribute to theprogression of pathological fibrosis and vascular remodeling (Lan T-H etal., Cardiovascular Pathology 22 (2013) 401-407).

It has been suggested that risk factors associated with cardiovasculardisease, including hypertension, hyperglycemia, dyslipidemia andhyperhomocysteinemia (HHcy), also act as initiation and progressionfactors of vascular fibrosis (Lan T-H et al., Cardiovascular Pathology22 (2013) 401-407).

8.1. Pathogenesis of Vascular Fibrosis

8.1.1. Renin-angiotensin-aldosterone system (RAAS)

RAAS has emerged as one of the essential links in the development ofvascular remodeling (Sun Y, Congest. Heart Fail. 2002; 8:11-6).Angiotensin II (Ang II) and aldosterone, the circulating effectorhormones of RAAS, are recognized as responsible for the pathophysiologyof vascular fibrosis.

Ang II, the principal effector hormone of the RAAS, not only mediatesimmediate physiological effects of vasoconstriction and blood pressureregulation, but also regulates many processes implicated in vascularpathophysiology, including cell growth/apoptosis of vascular cells,migration of VSMCs, inflammatory responses and ECM remodeling(Ruiz-Ortega M et al., Hypertension 2001; 38:1382-7). Ang II actsthrough two main specific receptors, AT1 and AT2. Binding of Ang II toAT1 receptor activates a series of signaling cascades, includingepidermal growth factor receptor (EGFR), platelet-derived growth factorreceptor (PDGFR), insulin receptor, c-Src family kinases, Ca2+-dependentproline-rich tyrosine kinase 2 (Pyk2), focal adhesion kinase (FAK) andJanus kinases (JAK), which in turn regulate the Ang II pathologiceffects in the vasculature (Hunyady L, Catt K J, Mol Endocrinol 2006;20: 953-70). AT1 also has been implicated in cell growth and hypertrophyby activating PKC and MAPKs, including ERK1/2, p38 MAPK, and c-JunNH2-terminal kinase (JNK) (Suzuki H et al., Curr Med Chem CardiovascHematol Agents 2005; 3:305-22). Ang II-mediated EGFR activation occursin a Src-dependent, redox-sensitive manner, and also bycalcium-dependent and -independent pathways (Eguchi S et al., J BiolChem 1998; 273:8890-6; Ushio-Fukai M et al., Arterioscler Thromb VascBiol 2001; 21:489-95). Evidence suggests that Ang II stimulates thephosphorylation of PDGF-β receptor (PDGFβ-R) through the binding oftyrosine-phosphorylated Shc to PDGFβ-R (Heeneman S et al., J Biol Chem2000; 275: 15926-32). Ang II also has been shown to increase serinephosphorylation of the insulin receptor β-subunit, causing insulinreceptor substrate (IRS-1) inactivation both by uncoupling it fromdownstream effectors (PI3K, PDK1, Akt) and by targeting it fordegradation in the proteasomal pathway (Folli F et al., J Clin Invest1997; 100: 2158-69; Natarajan R et al., Hypertension 1999; 33:378-84).Ang II promotes cellular proliferation and ECM synthesis and inductionof several mediators, such as TGF-beta, connective tissue growth factor(CTGF), cytokines (e.g., IL-6, TNF-alpha), and monocyte chemoattractantprotein type 1 (MCP-1). It is generally accepted that Ang II-induced ECMproduction is mainly mediated by up-regulation of TGF-β and itsdownstream mediator CTGF (Border W A, Noble N A, Hypertension 1998;31:181-8; Wolf G, Kidney Int 2006; 70:1914-9). Ang II activates thebinding of nuclear proteins to the binding site of the activatorprotein-1 (AP-1) of the TGF-β1 promoter through PKC and p38MAPK-dependent pathways. Recent studies have shown that Ang II alsoinduces CTGF expression via TGF-beta-independent Smad signaling pathwaysand increases the production of MMPs involved in matrix degradation,which leads to weakening of vessel walls (Rodriguez-Vita J et al.,Circulation 2005; 111:2509-17; Yang F et al., Hypertension 2009;54:877-84). Ang II is known to stimulate aldosterone production, thuspromoting fibrosis and collagen formation (Brilla C G et al., J Mol CellCardiol 1994; 26:809-20).

Aldosterone promotes fibrosis and collagen formation through severalmechanisms, including upregulation of Ang II receptors, stimulation ofTGF-β and CTGF synthesis, stimulation of MMPs activity, andparticipation in VSMC hypertrophy and endothelial dysfunction (EpsteinM, Nephrol Dial Transplant 2003; 18:1984-92). It has been reported thataldosterone stimulates the expression of plasminogen activatorinhibitor-1 (PAI-1), the major physiological inhibitor of plasminogenactivators, which is implicated in ECM accumulation by inhibiting matrixdegradation and inducing CTGF expression via both p38 MAPK cascade andmineralocorticoid receptor (Brown N J, et al., Kidney Int 2000;58:1219-27; Lee Y S et al., J Korean Med Sci 2004; 19: 805-11).Aldosterone has been shown to induce MMP activity in adult ratventricular myocytes via activation of the mineralocorticoid receptor,PKC, and reactive oxygen species (ROS)-dependent activation of theMEK/ERK pathway (Rude M K et al., Hypertension 2005; 46:555-61).

8.1.2. Transforming Growth Factor-β (TGF-β)

Transforming growth factor (TGF)-β is a ubiquitously expressed cytokinethat belongs to a large superfamily, of which TGF-β1 is most frequentlyup-regulated in ECM remodeling (Massague J et al., Cell 2000;103:295-309). TGF-β signals through a heteromeric cell-surface complexof two types of transmembrane serine/threonine kinases, type I receptorsand type II receptors (Derynck R et al., Nat Genet 2001; 29:117-2). TypeI receptor phosphorylation, which is activated by type II receptors, isessential for the activation of downstream target proteins (Derynck R,Feng X H, Biochim Biophys Acta 1997; 1333:F105-50).

TGF-β predominantly transmits signals through transcription factorscalled Smads (Massague J et al., Genes Dev 2005; 19: 2783-810.). Smad2and Smad3 are specific mediators of TGF-beta/activin pathways, whereasSmad1, Smad5 and Smad8 are involved in bone morphogenetic protein (BMP)signaling (especially BMP-7) which inhibits VSMC proliferation viaI-Smad (Smad6 and Smad7) activation (Singh N N, Ramji D P, CytokineGrowth Factor Rev 2006; 17:487-99). Studies have shown thatoverexpression of Smad6 may selectively inhibit BMP receptor signalingwhereas Smad7 inhibits both BMP and TGF-beta/activin receptor signaling(Nakao A et al., Trends Mol Med 2002; 8: 361-3). Smad7 expression,resulting in the inhibition of TGF-beta-induced fibronectin, collagenand CTGF expression, can also be induced by activation of the EGFreceptor, IL-gamma signaling through signal transducer and activator oftranscription (STAT), and TNF-alpha induced activation of NF-Kb (Wang Met al., Arterioscler Thromb Vasc Biol 2006; 26:1503-9; Ikedo H et al.,Int J Mol Med 2003; 11: 645-50.).

In addition to the Smad pathway, non-Smad pathways also participate inTGF-beta signaling and serve as nodes for crosstalk with other majorsignaling pathways (Ruiz-Ortega M et al., Cardiovasc Res 2007; 74:196-206; Bobik A, Arterioscler Thromb Vasc Biol 2006; 26: 1712-20). TheJNK/p38, Erk/MAPK, Rho-like GTPase, and PI3/Akt pathways are believed toreinforce, attenuate or modulate downstream cellular responses possiblyaccounting for the varying effects of TGF-β (Zhang F et al., J Biol Chem2009; 284:17564-74). TGF-β participates in the pathogenesis of manycardiovascular diseases, including hypertension, restenosis,atherosclerosis, cardiac hypertrophy, and heart failure. TGF-β plays acritical role in ECM accumulation and vascular remodeling viaup-regulating the production of several agents, including growth factors(e.g., CTGF, FGF), related genes (e.g., c-myc, cjun, junBJI, p53) andPAI-1 (Perbal B, Lancet 2004; 363: 62-4; Hayashida T et al., FASEB J2003; 17:1576-8; Samarakoon R et al., J Cell Physiol 2005; 204:236-46;Seay U et al., J Pharmacol Exp Ther 2005; 315:1005-12). Angiotensin II,mechanical stress, endothelin-I, high glucose, extremes of temperatureand pH, steroids, and reactive oxygen species have been found tostimulate TGF-β activation as a mediator of vascular fibrosis(Ruiz-Ortega M et al., Curr Hypertens Rep 2003; 5: 73-9; Li J H et al.,Kidney Int 2003; 63: 2010-9.). In addition, MMPs (such as MMP-2 and -9)enhance the release of TGF-β, which stimulates TIMP, ultimatelyresulting in the inhibition of ECM (i.e., ECM accumulation) and vascularremodeling (Derynck R, Zhang Y E, Nature 2003; 425:577-84).

8.1.3. Connective Tissue Growth Factor (CTGF)

CTGF, a potent pro-fibrotic growth factor, has been implicated infibroblast proliferation, cellular adhesion, angiogenesis and ECMsynthesis (Ruperez M et al., Circulation 2003; 108:1499-505). CTGFpromotes VSMC proliferation, migration, and production of ECM, which mayplay a role in the development and progression of atherosclerosis (LeaskA et al., Curr Rheumatol Rep 2002; 4: 136-42). CTGF expression isregulated by several agents, including TGF-β, TNF-α, cAMP, high glucose,dexamethasone, factor VIIa, and mechanical stress (Lau L F, Lam S C, ExpCell Res 1999; 248: 44-57). TGF-β-induced CTGF production is involvedwith several signal pathways, including Smads, Ras/MEK/Erk, Ap-1/JNK,PKC, and Tyr, and its expression can be decreased by TNF-α, cAMP, PGE2,IL-4 and PPAR through TGF-β down-regulation (Leask A et al., J Biol Chem2003; 278: 13008-15; Leask A, Abraham D J, Biochem Cell Biol 2003; 81:355-63). CTGF also appears to increase the expression of MMP-2 (Fan W H,Karnovsky M J, J Biol Chem 2002; 277: 9800-5). It has been reported thatthe addition of CTGF to primary mesangial cells induced fibronectinproduction, cell migration, and cytoskeletal rearrangement, which wereassociated with recruitment of Src and phosphorylation of p42/44 MAPKand protein kinase B (Crean J K et al, J Biol Chem 2002; 277:44187-94).

8.1.4. Matrix metalloproteinases (MMPs)

MMPs, together with cysteine proteinases, aspartic proteinases, andserine proteinases are proteolytic enzymes involved in ECM and basementmembranes (BMs) degradation (Lan T-H et al., Cardiovascular Pathology 22(2013) 401-407). MMPs play important roles in the physiology offibrosis, as in liver cirrhosis, fibrotic lung disease, otosclerosis,atherosclerosis, and multiple sclerosis. MMPs are thought to mediate theprogression of stable atherosclerotic lesions to an unstable phenotypethat is prone to rupture through the destruction of ECM proteins (LanT-H et al., Cardiovascular Pathology 22 (2013) 401-407). MMP-1, MMP-2,MMP-3 and MMP-9 participate in weakening the connective tissue matrix inthe intima, which leads to plaque rupture, acute thrombosis, and SMCproliferation and migration (Amalinei C et al., Rom J Morphol Embryol2010; 51: 215-28). MMP degradation of the EC basement membrane duringdiapedesis of inflammatory cells could contribute to a decreasedendothelial barrier function with increased influx of plasma proteins.All of these interactions have been shown to increase production of MMPsin macrophages, which may also provide stimuli for MMP production inneighboring cells and mechanisms for activation of secreted MMP zymogensresulting in developing atherosclerotic lesions that may facilitatefurther structural changes and enable their growth (Galis Z S, Khatri JJ, Circ Res 2002; 90: 251-62).

The expression of most MMPs is up-regulated during certain physiologicaland pathological remodeling processes, and mediated by a variety ofinflammatory cytokines, hormones, and growth factors, such as IL-1,IL-6, TNF-α, EGF, PDGF, basic fibroblast growth factor (bFGF), and CD40[111-113]. TGF-β up-regulates the expression of MMP-2 and MMP-9, whileit decreases the expression of MMP-1 and MMP-3 (Mauviel A, J CellBiochem 1993; 53: 288-95).

Fully activated MMPs can be inhibited by tissue inhibitors ofmetalloproteinases (TIMPs). An imbalance between myocardial MMPs andTIMPs results in collagen accumulation, adverse matrix remodeling andreactive interstitial fibrosis (Nagase H et al., Cardiovasc Res 2006;69: 562-73).

8.1.5. Peroxisome Proliferator-Activated Receptor Gamma (PPARγ)

It has been reported that aberrant expression or function of PPARγcontributes to the progression of pathological fibrosis and vascularremodeling (Wei J et al., Curr Opin Rheumatol 2010; 22: 671-6). Theantagonistic effects of PPARγ on abrogating TGF-β induced stimulation ofcollagen and fibronectin synthesis and the secretion of fibrotic growthfactors including TGF-β and CTGF revealed the anti-fibrotic role ofPPARγ in fibrogenesis (Ghosh A K et al., Arthritis Rheum 2004; 50:1305-18; Burgess H A et al., Am J Physiol Lung Cell Mol Physiol 2005;288: L1146-53). It was observed that PPARγ abrogates Smad-dependentcollagen stimulation by targeting the p300 transcriptional co-activator(Ghosh A K et al., FASEB J 2009; 23: 2968-77). It has been hypothesizedthat PPARγ-independent pathways might also be involved in theanti-fibrotic effects triggered by PPARγ ligands, including inhibitionof fibroblast migration, adipocyte differentiation and myofibroblasttransition (Lan T-H et al., Cardiovascular Pathology 22 (2013) 401-407).

9. Current and Emerging Therapeutic Approaches for Treating FibroticDiseases or Conditions

Therapeutic agents currently being used to treat fibrotic diseases aredisclosed in Datta et al., British Journal of Pharmacology, 163:141-172, 2011; incorporated by reference herein). Non-limiting examplesof such therapeutic agents include, but are not limited to, purifiedbovine Type V collagens (e.g., IW-001; ImmuneWorks; UnitedTherapeutics), IL-13 receptor antagonists (e.g., QAX576; Novartis),protein tyrosine kinase inhibitors (e.g., imatinib (Gleevec®); CraigDaniels/Novartis), endothelial receptor antagonists (e.g., ACT-064992(macitentan); Actelion), dual endothelin receptor antagonists (e.g.,bosentan (Tracleer®); Actelion), prostacyclin analogs (inhaled iloprost(e.g., Ventavis®); Actelion), anti-CTGF monoclonal antibodies (e.g.,FG-3019), endothelin receptor antagonists (A-selective) (e.g.,ambrisentan (Letairis®), Gilead), AB0024 (Arresto), lysyl oxidase-like 2(LOXL2) monoclonal antibodies (e.g., GS-6624 (formerly AB0024); Gilead),c-Jun N-terminal kinase (JNK) inhibitors (e.g., CC-930; Celgene),Pirfenidone (e.g., Esbriet® (InterMune), Pirespa® (Shionogi)), IFN-γ1b(e.g., Actimmune®; InterMune), pan-neutralizing IgG4 human antibodiesagainst all three TGF-β isoforms (e.g., GC1008; Genzyme), TGF-βactivation inhibitors (e.g., Stromedix (STX-100)) recombinant humanPentraxin-2 protein (rhPTX-2) (e.g., PRM151; Promedior), bispecificIL4/IL13 antibodies (e.g., SAR156597; Sanofi), humanized monoclonalantibodies targeting integrin αvβ6 (BIBF 1120; Boehringer Ingelheim),N-acetylcysteine (Zambon SpA), Sildenafil (Viagra®), TNF antagonists(e.g., etanercept (Enbrel®); Pfizer), glucocorticoids (e.g., prednisone,budesonide, mometasone furoate, fluticasone propionate, and fluticasonefuroate), bronchodilators (e.g., leukotriene modifers (e.g., Montelukast(SINGUAIR®)), anticholingertic bronchodilators (e.g., Ipratropiumbromide and Tiotropium), short-acting β2-agonists (e.g., isoetharinemesylate (Bronkometer®), adrenalin, salbutanol/albuterol, andterbutaline), long-acting β2-agonists (e.g., salmeterol, formoterol,indecaterol (Onbrez®), and combination bronchodilators including, butnot limited to, SYMBICORT® (containing both budesonide and formoterol),corticosteroids (e.g., prednisone, budesonide, mometasone furoate),methylated xanthine and its derivatives (e.g., caffeine, aminophylline,IBMX, paraxanthine, pentoxifylline, theobromine, and theophylline),neutrophil elastase inhibitors (e.g., ONO-5046, MR-889, L-694,458,CE-1037, GW-311616, and TEI-8362, and transition-state inhibitors, suchas ONO-6818, AE-3763, FK-706, ICI-200,880, ZD-0892 and ZD-8321),phosphodiesterase inhibitors (e.g., roflumilast (DAXAS®; Daliresp®),cilomilast (Ariflo®, SB-207499) and sofosbuvir (Sovaldil®).

9.1. Current Therapies for Liver Fibrosis

Despite significant advances in understanding hepatic fibrosis and indefining targets for therapy, a limited number of anti-fibrotic drugsare approved for clinical use in patients with advanced liver disease(Mormone E. et al., Chem. Biol. Interact. 2011 Sep. 30; 193(3):225-231). Regression of established fibrosis can be accomplished inselected individuals with chronic liver diseases subjected to effectiveinterferon therapies (Shiratori Y, et al., Ann. Intern. Med. 2000;132(7): 517-524). However, a large cohort of patients does not respondto conventional treatment and thus remain at risk for progression offibrosis to cirrhosis (Mormone E. et al., Chem. Biol. Interact. 2011Sep. 30; 193(3): 225-231).

Several compounds with potential antifibrotic activity, includingcolchicine and malotilate, have been studied in human trials, but werefound not effective (Brenner D A and Alcorn J M, Semin. Liver Dis. 1990;10(1): 75-83; Takase S, et al., Gastroenterol. Jpn., 1988; 23(6):639-645). Many agents such as malotilate, genistein, curcumin andsilymarin have been shown to be effective in vitro and in experimentalanimal models (Takase S, et al., Gastroenterol Jpn. 1988; 23(6):639-645; Fu Y M, et al., Mol. Pharm., 2008; 3(2): 399-409; George J, etal., Biomedicine, 2006; 26(3-4): 18-26). Combination therapy that worksat different mechanistic levels may be more appropriate to block HSCactivation and the pathogenesis of liver fibrosis (Mormone E. et al.,Chem. Biol. Interact. 2011 Sep. 30; 193(3): 225-231).

10. Kinases and Phosphorylation

Kinases are a ubiquitous group of enzymes that catalyze the phosphoryltransfer reaction from a phosphate donor (usuallyadenosine-5′-triphosphate (ATP)) to a receptor substrate. Although allkinases catalyze essentially the same phosphoryl transfer reaction, theydisplay remarkable diversity in their substrate specificity, structure,and the pathways in which they participate. A recent classification ofall available kinase sequences (approximately 60,000 sequences)indicates kinases can be grouped into 25 families of homologous (meaningderived from a common ancestor) proteins. These kinase families areassembled into 12 fold groups based on similarity of structural fold.Further, 22 of the 25 families (approximately 98.8% of all sequences)belong to 10 fold groups for which the structural fold is known. Of theother 3 families, polyphosphate kinase forms a distinct fold group, andthe 2 remaining families are both integral membrane kinases and comprisethe final fold group. These fold groups not only include some of themost widely spread protein folds, such as Rossmann-like fold (three ormore parallel β strands linked by two a helices in the topological orderβ-α-β-α-β), ferredoxin-like fold (a common α+β protein fold with asignature βαβα secondary structure along its backbone), TIM-barrel fold(meaning a conserved protein fold consisting of eight α-helices andeight parallel β-strands that alternate along the peptide backbone), andantiparallel β-barrel fold (a beta barrel is a large beta-sheet thattwists and coils to form a closed structure in which the first strand ishydrogen bonded to the last), but also all major classes (all α, all β,α+β, α/β) of protein structures. Within a fold group, the core of thenucleotide-binding domain of each family has the same architecture, andthe topology of the protein core is either identical or related bycircular permutation. Homology between the families within a fold groupis not implied.

Group I (23,124 sequences) kinases incorporate protein S/T-Y kinase,atypical protein kinase, lipid kinase, and ATP grasp enzymes and furthercomprise the protein S/T-Y kinase, and atypical protein kinase family(22,074 sequences). These kinases include: choline kinase (EC 2.7.1.32);protein kinase (EC 2.7.137); phosphorylase kinase (EC 2.7.1.38);homoserine kinase (EC 2.7.1.39); I-phosphatidylinositol 4-kinase (EC2.7.1.67); streptomycin 6-kinase (EC 2.7.1.72); ethanolamine kinase (EC2.7.1.82); streptomycin 3′-kinase (EC 2.7.1.87); kanamycin kinase (EC2.7.1.95); 5-methylthioribose kinase (EC 2.7.1.100); viomycin kinase (EC2.7.1.103); [hydroxymethylglutaryl-CoA reductase (NADPH2)] kinase (EC2.7.1.109); protein-tyrosine kinase (EC 2.7.1.112); [isocitratedehydrogenase (NADP+)] kinase (EC 2.7.1.116); [myosin light-chain]kinase (EC 2.7.1.117); hygromycin-B kinase (EC 2.7.1.119);calcium/calmodulin-dependent protein kinase (EC 2.7.1.123); rhodopsinkinase (EC 2.7.1.125); [beta-adrenergic-receptor] kinase (EC 2.7.1.126);[myosin heavy-chain] kinase (EC 2.7.1.129); [Tau protein] kinase (EC2.7.1.135); macrolide 2′-kinase (EC 2.7.1.136); I-phosphatidylinositol3-kinase (EC 2.7.1.137); [RNA-polymerase]-subunit kinase (EC 2.7.1.141);phosphatidylinositol-4,5-bisphosphate 3-kinase (EC 2.7.1.153); andphosphatidylinositol-4-phosphate 3-kinase (EC 2.7.1.154). Group Ifurther comprises the lipid kinase family (321 sequences). These kinasesinclude: I-phosphatidylinositol-4-phosphate 5-kinase (EC 2.7.1.68); ID-myo-inositol-triphosphate 3-kinase (EC 2.7.1.127);inositol-tetrakisphosphate 5-kinase (EC 2.7.1.140);I-phosphatidylinositol-5-phosphate 4-kinase (EC 2.7.1.149);I-phosphatidylinositol-3-phosphate 5-kinase (EC 2.7.1.150);inositol-polyphosphate multikinase (EC 2.7.1.151); andinositol-hexakiphosphate kinase (EC 2.7.4.21). Group I further comprisesthe ATP-grasp kinases (729 sequences) which includeinositol-tetrakisphosphate I-kinase (EC 2.7.1.134); pyruvate, phosphatedikinase (EC 2.7.9.1); and pyruvate, water dikinase (EC 2.7.9.2).

Group II (17,071 sequences) kinases incorporate the Rossman-likekinases. Group II comprises the P-loop kinase family (7,732 sequences).These include gluconokinase (EC 2.7.1.12); phosphoribulokinase (EC2.7.1.19); thymidine kinase (EC 2.7.1.21); ribosylnicotinamide kinase(EC 2.7.1.22); dephospho-CoA kinase (EC 2.7.1.24); adenylylsulfatekinase (EC 2.7.1.25); pantothenate kinase (EC 2.7.1.33); protein kinase(bacterial) (EC 2.7.1.37); uridine kinase (EC 2.7.1.48); shikimatekinase (EC 2.7.1.71); deoxycytidine kinase (EC 2.7.1.74); deoxyadenosinekinase (EC 2.7.1.76); polynucleotide 5′-hydroxyl-kinase (EC 2.7.1.78);6-phosphofructo-2-kinase (EC 2.7.1.105); deoxyguanosine kinase (EC2.7.1.113); tetraacyldisaccharide 4′-kinase (EC 2.7.1.130);deoxynucleoside kinase (EC 2.7.1.145); adenosylcobinamide kinase (EC2.7.1.156); polyphosphate kinase (EC 2.7.4.1); phosphomevalonate kinase(EC 2.7.4.2); adenylate kinase (EC 2.7.4.3); nucleoside-phosphate kinase(EC 2.7.4.4); guanylate kinase (EC 2.7.4.8); thymidylate kinase (EC2.7.4.9); nucleoside-triphosphate-adenylate kinase (EC 2.7.4.10);(deoxy)nucleoside-phosphate kinase (EC 2.7.4.13); cytidylate kinase (EC2.7.4.14); and uridylate kinase (EC 2.7.4.22). Group II furthercomprises the phosphoenolpyruvate carboxykinase family (815 sequences).These enzymes include protein kinase (HPr kinase/phosphatase) (EC2.7.1.37); phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32); andphosphoenolpyruvate carboxykinase (ATP) (EC 4.1.1.49). Group II furthercomprises the phosphoglycerate kinase (1,351 sequences) family. Theseenzymes include phosphoglycerate kinase (EC 2.7.2.3) andphosphoglycerate kinase (GTP) (EC 2.7.2.10). Group II further comprisesthe aspartokinase family (2,171 sequences). These enzymes includecarbamate kinase (EC 2.7.2.2); aspartate kinase (EC 2.7.2.4);acetylglutamate kinase (EC 2.7.2.8 1); glutamate 5-kinase (EC 2.7.2.1)and uridylate kinase (EC 2.7.4.). Group II further comprises thephosphofructokinase-like kinase family (1,998 sequences). These enzymesinclude 6-phosphofrutokinase (EC 2.7.1.11); NAD (+) kinase (EC2.7.1.23); I-phosphofructokinase (EC 2.7.1.56);diphosphate-fructose-6-phosphate I-phosphotransferase (EC 2.7.1.90);sphinganine kinase (EC 2.7.1.91); diacylglycerol kinase (EC 2.7.1.107);and ceramide kinase (EC 2.7.1.138). Group II further comprises theribokinase-like family (2,722 sequences). These enzymes include:glucokinase (EC 2.7.1.2); ketohexokinase (EC 2.7.1.3); fructokinase (EC2.7.1.4); 6-phosphofructokinase (EC 2.7.1.11); ribokinase (EC 2.7.1.15);adenosine kinase (EC 2.7.1.20); pyridoxal kinase (EC 2.7.1.35);2-dehydro-3-deoxygluconokinase (EC 2.7.1.45); hydroxymethylpyrimidinekinase (EC 2.7.1.49); hydroxyethylthiazole kinase (EC 2.7.1.50);I-phosphofructokinase (EC 2.7.1.56); inosine kinase (EC 2.7.1.73);5-dehydro-2-deoxygluconokinase (EC 2.7.1.92); tagatose-6-phosphatekinase (EC 2.7.1.144); ADP-dependent phosphofructokinase (EC 2.7.1.146);ADP-dependent glucokinase (EC 2.7.1.147); and phosphomethylpyrimidinekinase (EC 2.7.4.7). Group II further comprises the thiaminpyrophosphokinase family (175 sequences) which includes thiaminpyrophosphokinase (EC 2.7.6.2). Group II further comprises the glyceratekinase family (107 sequences) which includes glycerate kinase (EC2.7.1.31).

Group III kinases (10,973 sequences) comprise the ferredoxin-like foldkinases. Group III further comprises the nucleoside-diphosphate kinasefamily (923 sequences). These enzymes include nucleoside-diphosphatekinase (EC 2.7.4.6). Group III further comprises the HPPK kinase family(609 sequences). These enzymes include2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (EC2.7.6.3). Group III further comprises the guanido kinase family (324sequences). These enzymes include guanidoacetate kinase (EC 2.7.3.1);creatine kinase (EC 2.7.3.2); arginine kinase (EC 2.7.3.3); andlombricine kinase (EC 2.7.3.5). Group III further comprises thehistidine kinase family (9,117 sequences). These enzymes include proteinkinase (histidine kinase) (EC 2.7.1.37); [pyruvate dehydrogenase(lipoamide)] kinase (EC 2.7.1.99); and [3-methyl-2-oxybutanoatedehydrogenase (lipoamide)] kinase (EC 2.7.1.115).

Group IV kinases (2,768 sequences) incorporate ribonuclease H-likekinases. These enzymes include hexokinase (EC 2.7.1.1); glucokinase (EC2.7.1.2); fructokinase (EC 2.7.1.4); rhamnulokinase (EC 2.7.1.5);mannokinase (EC 2.7.1.7); gluconokinase (EC 2.7.1.12); L-ribulokinase(EC 2.7.1.16); xylulokinase (EC 2.7.1.17); erythritol kinase (EC2.7.1.27); glycerol kinase (EC 2.7.1.30); pantothenate kinase (EC2.7.1.33); D-ribulokinase (EC 2.7.1.47); L-fucolokinase (EC 2.7.1.51);L-xylulokinase (EC 2.7.1.53); allose kinase (EC 2.7.1.55);2-dehydro-3-deoxygalactonokinase (EC 2.7.1.58); N-acetylglucosaminekinase (EC 2.7.1.59); N-acylmannosamine kinase (EC 2.7.1.60);polyphosphate-glucose phosphotransferase (EC 2.7.1.63); beta-glucosidekinase (EC 2.7.1.85); acetate kinase (EC 2.7.2.1); butyrate kinase (EC2.7.2.7); branched-chain-fatty-acid kinase (EC 2.7.2.14); and propionatekinase (EC 2.7.2.15).

Group V kinases (1,119 sequences) incorporate TIM β-barrel kinases.These enzymes include pyruvate kinase (EC 2.7.1.40).

Group VI kinases (885 sequences) incorporate GHMP kinases. These enzymesinclude galactokinase (EC 2.7.1.6); mevalonate kinase (EC 2.7.1.36);homoserine kinase (EC 2.7.1.39); L-arabinokinase (EC 2.7.1.46);fucokinase (EC 2.7.1.52); shikimate kinase (EC 2.7.1.71); 4-(cytidine5′-diphospho)-2-C-methyl-D-erythriol kinase (EC 2.7.1.148); andphosphomevalonate kinase (EC 2.7.4.2).

Group VII kinases (1,843 sequences) incorporate AIR synthetase-likekinases. These enzymes include thiamine-phosphate kinase (EC 2.7.4.16)and selenide, water dikinase (EC 2.7.9.3).

Group VIII kinases (565 sequences) incorporate riboflavin kinases (565sequences). These enzymes include riboflavin kinase (EC 2.7.1.26).

Group IX kinases (197 sequences) incorporate dihydroxyacetone kinases.These enzymes include glycerone kinase (EC 2.7.1.29).

Group X kinases (148 sequences) incorporate putative glycerate kinases.These enzymes include glycerate kinase (EC 2.7.1.31).

Group XI kinases (446 sequences) incorporate polyphosphate kinases.These enzymes include polyphosphate kinases (EC 2.7.4.1).

Group XII kinases (263 sequences) incorporate integral membrane kinases.Group XII comprises the dolichol kinase family. These enzymes includedolichol kinases (EC 2.7.1.108). Group XII further comprises theundecaprenol kinase family. These enzymes include undecaprenol kinases(EC 2.7.1.66).

Kinases play indispensable roles in numerous cellular metabolic andsignaling pathways, and they are among the best-studied enzymes at thestructural level, biochemical level, and cellular level. Despite thefact that all kinases use the same phosphate donor (in most cases, ATP)and catalyze apparently the same phosphoryl transfer reaction, theydisplay remarkable diversity in their structural folds and substraterecognition mechanisms. This probably is due largely to theextraordinary diverse nature of the structures and properties of theirsubstrates.

10.1. Mitogen-Activated Protein Kinase-Activated Protein Kinases (MK2and MK3)

Different groups of MAPK-activated protein kinases (MAP-KAPKs) have beendefined downstream of mitogen-activated protein kinases (MAPKs). Theseenzymes transduce signals to target proteins that are not directsubstrates of the MAPKs and, therefore, serve to relayphosphorylation-dependent signaling with MAPK cascades to diversecellular functions. One of these groups is formed by the three MAPKAPKs:MK2, MK3 (also known as 3pK), and MK5 (also designated PRAK).Mitogen-activated protein kinase-activated protein kinase 2 (alsoreferred to as “MAPKAPK2”, “MAPKAP-K2”, or “MK2”) is a kinase of theserine/threonine (Ser/Thr) protein kinase family. MK2 is highlyhomologous to MK3 (approximately 75% amino acid identity). The kinasedomains of MK2 and MK3 are most similar (approximately 35% to 40%identity) to calcium/calmodulin-dependent protein kinase (CaMK),phosphorylase b kinase, and the C-terminal kinase domain (CTKD) of theribosomal S6 kinase (RSK) isoforms. The mk2 gene encodes twoalternatively spliced transcripts of 370 amino acids (MK2A) and 400amino acids (MK2B). The mk3 gene encodes one transcript of 382 aminoacids. The MK2- and MK3 proteins are highly homologous, yet MK2Apossesses a shorter C-terminal region. The C-terminus of MK2B contains afunctional bipartite nuclear localization sequence (NLS)(Lys-Lys-Xaa₁₀-Lys-Arg-Arg-Lys-Lys; SEQ ID NO: 23) that is not presentin the shorter MK2A isoform, indicating that alternative splicingdetermines the cellular localization of the MK2 isoforms. MK3 possessesa similar nuclear localization sequence. The nuclear localizationsequence found in both MK2B and MK3 encompasses a D domain(Leu-Leu-Lys-Arg-Arg-Lys-Lys; SEQ ID NO: 24) that studies have shown tomediate the specific interaction of MK2B and MK3 with p38α and p38β.MK2B and MK3 also possess a functional nuclear export signal (NES)located N-terminal to the NLS and D domain. The NES in MK2B issufficient to trigger nuclear export following stimulation, a processwhich may be inhibited by leptomycin B. The sequence N-terminal to thecatalytic domain in MK2 and MK3 is proline rich and contains one (MK3)or two (MK2) putative Src homology 3 (SH3) domain-binding sites, whichstudies have shown, for MK2, to mediate binding to the SH3 domain ofc-Abl in vitro. Recent studies suggest that this domain is involved inMK2-mediated cell migration.

MK2B and MK3 are located predominantly in the nucleus of quiescent cellswhile MK2A is present in the cytoplasm. Both MK2B and MK3 are rapidlyexported to the cytoplasm via a chromosome region maintenance protein(CRM1)-dependent mechanism upon stress stimulation. Nuclear export ofMK2B appears to be mediated by kinase activation, as phosphomimeticmutation of Thr334 within the activation loop of the kinase enhances thecytoplasmic localization of MK2B. Without being limited by theory, it isthought that MK2B and MK3 may contain a constitutively active NLS and aphosphorylation-regulated NES.

MK2 and MK3 appear to be expressed ubiquitously, with predominantexpression in the heart, in skeletal muscle, and in kidney tissues.

10.1.1. Activation

Various activators of p38α and p38β potently stimulate MK2 and MK3activity. p38 mediates the in vitro and in vivo phosphorylation of MK2on four proline-directed sites: Thr25, Thr222, Ser272, and Thr334. Ofthese sites, only Thr25 is not conserved in MK3. Without being limitedby theory, while the function of phosphorylated Thr25 in unknown, itslocation between the two SH3 domain-binding sites suggests that it mayregulate protein-protein interactions. Thr222 in MK2 (Thr201 in MK3) islocated in the activation loop of the kinase domain and has been shownto be essential for MK2 and MK3 kinase activity. Thr334 in MK2 (Thr313in MK3) is located C-terminal to the catalytic domain and is essentialfor kinase activity. The crystal structure of MK2 has been resolved and,without being limited by theory, suggests that Thr334 phosphorylationmay serve as a switch for MK2 nuclear import and export. Phosphorylationof Thr334 also may weaken or interrupt binding of the C terminus of MK2to the catalytic domain, exposing the NES and promoting nuclear export.

Studies have shown that, while p38 is capable of activating MK2 and MK3in the nucleus, experimental evidence suggests that activation andnuclear export of MK2 and MK3 are coupled by a phosphorylation-dependentconformational switch that also dictates p38 stabilization andlocalization, and the cellular location of p38 itself is controlled byMK2 and possibly MK3. Additional studies have shown that nuclear p38 isexported to the cytoplasm in a complex with MK2 followingphosphorylation and activation of MK2. The interaction between p38 andMK2 may be important for p38 stabilization since studies indicate thatp38 levels are low in MK2-deficient cells and expression of acatalytically inactive MK2 protein restores p38 levels.

10.1.2. Substrates and Functions

Further studies have shown that the small heat shock protein HSPB1 (alsoknown as heat shock protein 27 or Hsp27), lymphocyte-specific proteinLSP-1, and vimentin are phosphorylated by MK2. HSPB1 is of particularinterest because it forms large oligomers, which may act as molecularchaperones and protect cells from heat shock and oxidative stress. Uponphosphorylation, HSPB1 loses its ability to form large oligomers and isunable to block actin polymerization, suggesting that MK2-mediatedphosphorylation of HSPB1 serves a homeostatic function aimed atregulating actin dynamics that otherwise would be destabilized duringstress.

MK3 also was shown to phosphorylate HSPB1 in vitro and in vivo, but itsrole during stressful conditions has not yet been elucidated. MK2 sharesmany substrates with MK3. Both enzymes have comparable substratepreferences and phosphorylate peptide substrates with similar kineticconstants. The minimum sequence required for efficient phosphorylationby MK2 was found to be Hyd-Xaa-Arg-Xaa-Xaa-pSer/Thr (SEQ ID NO: 25),where Hyd is a bulky hydrophobic residue.

Experimental evidence supports a role for p38 in the regulation ofcytokine biosynthesis and cell migration. The targeted deletion of themk2 gene in mice suggested that although p38 mediates the activation ofmany similar kinases, MK2 seems to be the key kinase responsible forthese p38-dependent biological processes. Loss of MK2 leads (i) to adefect in lipopolysaccharide (LPS)-induced synthesis of cytokines suchas tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and gammainterferon (IFN-γ) and (ii) to changes in the migration of mouseembryonic fibroblasts, smooth muscle cells, and neutrophils.

Consistent with a role for MK2 in inflammatory responses, MK2-deficientmice showed increased susceptibility to Listeria monocytogenes infectionand reduced inflammation-mediated neuronal death following focalischemia. Since the levels of p38 protein also are reduced significantlyin MK2-deficient cells, it was necessary to distinguish whether thesephenotypes were due solely to the loss of MK2. To achieve this, MK2mutants were expressed in MK2-deficient cells, and the results indicatedthat the catalytic activity of MK2 was not necessary to restore p38levels but was required to regulate cytokine biosynthesis.

The knockout or knockdown studies of MK2 provided strong support thatactivated MK2 enhances stability of IL-6 mRNA through phosphorylation ofproteins interacting with the AU-rich 3′ untranslated region of IL-6mRNA. In particular, it has been shown that MK2 is principallyresponsible for phosphorylation of hnRNPA0, an mRNA-binding protein thatstabilizes IL-6 RNA. In addition, several additional studiesinvestigating diverse inflammatory diseases have found that levels ofpro-inflammatory cytokines, such as IL-6, IL-1β, TNF-α and IL-8, areincreased in induced sputum from patients with stable chronicobstructive pulmonary disease (COPD) or from the alveolar macrophages ofcigarette smokers (Keatings V. et al, Am J Resp Crit Care Med, 1996,153:530-534; Lim, S. et al., J Respir Crit Care Med, 2000,162:1355-1360). Elevated levels of pro-inflammatory cytokines, such asinterleukin-8 (IL-8) and interleukin-6 (IL-6), as well as relateddownstream cell adhesion molecules (CAMs) such as intercellular adhesionmolecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1),matrix metalloproteinases such as matrix metalloproteinase-7 (MMP-7),and signaling molecules such as 5100 calcium-binding protein A12(S100A12, also known as calgranulin C), in the peripheral blood havebeen found to be associated with mortality, lung transplant-freesurvival, and disease progression in patients with idiopathic pulmonaryfibrosis (Richards et al., Am J Respir Crit Care Med, 2012, 185: 67-76;Richards, T. et al., Am J Respir Crit Care Med, 181: A1120, 2010;Moodley, Y. et al., Am J Respir Cell Mol Biol., 29(4): 490-498, 2003).Taken together, these studies implicate that elevated levels ofinflammatory cytokines induced by MK2 activation may be involved in thepathogenesis of airway or lung tissue diseases; and suggest a potentialfor anti-cytokine therapy for treating airway or lung tissue diseases,such as idiopathic pulmonary fibrosis and chronic obstructive pulmonarydisease (COPD) (Chung, K., Eur Respir J, 2001, 18: Suppl. 34: 50-59).

10.1.3. Regulation of mRNA Translation

Previous studies using MK2 knockout mice or MK2-deficient cells haveshown that MK2 increases the production of inflammatory cytokines,including TNF-α, IL-1, and IL-6, by increasing the rate of translationof its mRNA. No significant reductions in the transcription, processing,and shedding of TNF-α could be detected in MK2-deficient mice. The p38pathway is known to play an important role in regulating mRNA stability,and MK2 represents a likely target by which p38 mediates this function.Studies utilizing MK2-deficient mice indicated that the catalyticactivity of MK2 is necessary for its effects on cytokine production andmigration, suggesting that, without being limited by theory, MK2phosphorylates targets involved in mRNA stability. Consistent with this,MK2 has been shown to bind and/or phosphorylate the heterogeneousnuclear ribonucleoprotein (hnRNP) A0, tristetraprolin, the poly(A)-binding protein PABP1, and HuR, a ubiquitously expressed member ofthe elav (embryonic-lethal abnormal visual in Drosophila melanogaster)family of RNA-binding protein. These substrates are known to bind orcopurify with mRNAs that contain AU-rich elements in the 3′ untranslatedregion, suggesting that MK2 may regulate the stability of AU-rich mRNAssuch as TNF-α. It currently is unknown whether MK3 plays similarfunctions, but LPS treatment of MK2-deficient fibroblasts completelyabolished hnRNP AO phosphorylation, suggesting that MK3 is not able tocompensate for the loss of MK2.

MK3 participates with MK2 in phosphorylation of the eukaryoticelongation factor 2 (eEF2) kinase. eEF2 kinase phosphorylates andinactivates eEF2. eEF2 activity is critical for the elongation of mRNAduring translation, and phosphorylation of eEF2 on Thr56 results in thetermination of mRNA translation. MK2 and MK3 phosphorylation of eEF2kinase on Ser377 suggests that these enzymes may modulate eEF2 kinaseactivity and thereby regulate mRNA translation elongation.

10.1.4. Transcriptional Regulation by MK2 and MK3

Nuclear MK2, similar to many MKs, contributes to the phosphorylation ofcAMP response element binding (CREB), serum response factor (SRF), andtranscription factor ER81. Comparison of wild-type and MK2-deficientcells revealed that MK2 is the major SRF kinase induced by stress,suggesting a role for MK2 in the stress-mediated immediate-earlyresponse. Both MK2 and MK3 interact with basic helix-loop-helixtranscription factor E47 in vivo and phosphorylate E47 in vitro.MK2-mediated phosphorylation of E47 was found to repress thetranscriptional activity of E47 and thereby inhibit E47-dependent geneexpression, suggesting that MK2 and MK3 may regulate tissue-specificgene expression and cell differentiation.

10.1.5. Other Targets of MK2 and MK3.

Several other MK2 and MK3 substrates also have been identified,reflective of the diverse functions of MK2 and MK3 in several biologicalprocesses. The scaffolding protein 14-3-3ζ is a physiological MK2substrate. Studies indicate 14-3-3ζ interacts with a number ofcomponents of cell signaling pathways, including protein kinases,phosphatases, and transcription factors. Additional studies have shownthat MK2-mediated phosphorylation of 14-3-3ζ on Ser58 compromises itsbinding activity, suggesting that MK2 may affect the regulation ofseveral signaling molecules normally regulated by 14-3-3ζ.

Additional studies have shown that MK2 also interacts with andphosphorylates the p16 subunit of the seven-member Arp2 and Arp3 complex(p16-Arc) on Ser77. p16-Arc has roles in regulating the actincytoskeleton, suggesting that MK2 may be involved in this process.

MK2 and MK3 also may phosphorylate 5-lipoxygenase. 5-lipoxygenasecatalyzes the initial steps in the formation of the inflammatorymediator leukotrienes. Tyrosine hydroxylase, glycogen synthase, and Aktalso were shown to be phosphorylated by MK2. Finally, MK2 phosphorylatesthe tumor suppressor protein tuberin on Ser1210, creating a docking sitefor 14-3-3ζ. Tuberin and hamartin normally form a functional complexthat negatively regulates cell growth by antagonizing mTOR-dependentsignaling, suggesting that p38-mediated activation of MK2 may regulatecell growth by increasing 14-3-3ζ binding to tuberin.

10.2. Kinase Inhibition

The eukaryotic protein kinases constitute one of the largestsuperfamilies of homologous proteins that are related by virtue of theircatalytic domains. Most related protein kinases are specific for eitherserine/threonine or tyrosine phosphorylation. Protein kinases play anintegral role in the cellular response to extracellular stimuli. Thus,stimulation of protein kinases is considered to be one of the mostcommon activation mechanisms in signal transduction systems. Manysubstrates are known to undergo phosphorylation by multiple proteinkinases, and a considerable amount of information on primary sequence ofthe catalytic domains of various protein kinases has been published.These sequences share a large number of residues involved in ATPbinding, catalysis, and maintenance of structural integrity. Mostprotein kinases possess a well conserved 30-32 kDa catalytic domain.

Studies have attempted to identify and utilize regulatory elements ofprotein kinases. These regulatory elements include inhibitors,antibodies, and blocking peptides.

10.2.1. Inhibitors

Enzyme inhibitors are molecules that bind to enzymes thereby decreasingenzyme activity. The binding of an inhibitor may stop a substrate fromentering the active site of the enzyme and/or hinder the enzyme fromcatalyzing its reaction. Inhibitor binding is either reversible orirreversible. Irreversible inhibitors usually react with the enzyme andchange it chemically (e.g., by modifying key amino acid residues neededfor enzymatic activity) so that it no longer is capable of catalyzingits reaction. In contrast, reversible inhibitors bind non-covalently anddifferent types of inhibition are produced depending on whether theseinhibitors bind the enzyme, the enzyme-substrate complex, or both.

Enzyme inhibitors often are evaluated by their specificity and potency.The term “specificity” as used in this context refers to the selectiveattachment of an inhibitor or its lack of binding to other proteins. Theterm “potency” as used herein refers to an inhibitor's dissociationconstant, which indicates the concentration of inhibitor needed toinhibit an enzyme.

Inhibitors of protein kinases have been studied for use as a tool inprotein kinase activity regulation. Inhibitors have been studied for usewith, for example, cyclin-dependent (Cdk) kinase, MAP kinase,serine/threonine kinase, Src Family protein tyrosine kinase, tyrosinekinase, calmodulin (CaM) kinase, casein kinase, checkpoint kinase(Chkl), glycogen synthase kinase 3 (GSK-3), c-Jun N-terminal kinase(JNK), mitogen-activated protein kinase 1 (MEK), myosin light chainkinase (MLCK), protein kinase A, Akt (protein kinase B), protein kinaseC, protein kinase G, protein tyrosine kinase, Raf kinase, and Rhokinase.

10.2.2. Blocking Peptides

A peptide is a chemical compound that is composed of a chain of two ormore amino acids whereby the carboxyl group of one amino acid in thechain is linked to the amino group of the other via a peptide bond.Peptides have been used inter alia in the study of protein structure andfunction. Synthetic peptides may be used inter alia as probes to seewhere protein-peptide interactions occur. Inhibitory peptides may beused inter alia in clinical research to examine the effects of peptideson the inhibition of protein kinases, cancer proteins and otherdisorders.

The use of several blocking peptides has been studied. For example,extracellular signal-regulated kinase (ERK), a MAPK protein kinase, isessential for cellular proliferation and differentiation. The activationof MAPKs requires a cascade mechanism whereby MAPK is phosphorylated byan upstream MAPKK (MEK) which then, in turn, is phosphorylated by athird kinase MAPKKK (MEKK). The ERK inhibitory peptide functions as aMEK decoy by binding to ERK.

Other blocking peptides include autocamtide-2 related inhibitory peptide(AIP). This synthetic peptide is a highly specific and potent inhibitorof Ca²⁺/calmodulin-dependent protein kinase II (CaMKII). AIP is anon-phosphorylatable analog of autocamtide-2, a highly selective peptidesubstrate for CaMKII. AIP inhibits CaMKII with an IC₅₀ of 100 nM (IC₅₀is the concentration of an inhibitor required to obtain 50% inhibition).The AIP inhibition is non-competitive with respect to syntide-2 (CaMKIIpeptide substrate) and ATP but competitive with respect toautocamtide-2. The inhibition is unaffected by the presence or absenceof Ca²⁺/calmodulin. CaMKII activity is inhibited completely by AIP (1μM) while PKA, PKC and CaMKIV are not affected.

Other blocking peptides include cell division protein kinase 5 (Cdk5)inhibitory peptide (CIP). Cdk5 phosphorylates the microtubule proteintau at Alzheimer's Disease-specific phospho-epitopes when it associateswith p25. p25 is a truncated activator, which is produced from thephysiological Cdk5 activator p35 upon exposure to amyloid β peptides.Upon neuronal infections with CIP, CIPs selectively inhibit p25/Cdk5activity and suppress the aberrant tau phosphorylation in corticalneurons. The reasons for the specificity demonstrated by CIP are notfully understood.

Additional blocking peptides have been studied forextracellular-regulated kinase 2 (ERK2), ERK3, p38/HOG1, protein kinaseC, casein kinase II, Ca²⁺/calmodulin kinase IV, casein kinase II, Cdk4,Cdk5, DNA-dependent protein kinase (DNA-PK), serine/threonine-proteinkinase PAK3, phosphoinositide (PI)-3 kinase, PI-5 kinase, PSTAIRE (thecdk highly conserved sequence), ribosomal S6 kinase, GSK-4, germinalcenter kinase (GCK), SAPK (stress-activated protein kinase), SEK1(stress signaling kinase), and focal adhesion kinase (FAK).

11. Cell Penetrating Peptides (CPPs)

Cell penetrating peptides (CPPs) are a class of peptides capable ofpenetrating the plasma membrane of mammalian cells and of transportingcompounds of many types and molecular weights across the membrane. Thesecompounds include effector molecules, such as proteins, DNA, conjugatedpeptides, oligonucleotides, and small particles such as liposomes. WhenCPPs are chemically linked or fused to other proteins, the resultingfusion proteins still are able to enter cells. Although the exactmechanism of transduction is unknown, internalization of these proteinsis not believed to be receptor-mediated or transporter-mediated. CPPsare generally 10-16 amino acids in length and may be grouped accordingto their composition, such as, for example, peptides rich in arginineand/or lysine.

The use of CPPs capable of transporting effector molecules into cellshas become increasingly attractive in the design of drugs as theypromote the cellular uptake of cargo molecules. These cell-penetratingpeptides, generally categorized as amphipathic (meaning having both apolar and a nonpolar end) or cationic (meaning of or relating tocontaining net positively charged atoms) depending on their sequence,provide a non-invasive delivery technology for macromolecules. CPPsoften are referred to as “Trojan peptides,” “membrane translocatingsequences,” “protein transduction domains (PTDs),” or “cell permeableproteins (CPPs).” CPPs also may be used to assist novel HSPB1 kinaseinhibitors to penetrate cell membranes. (see U.S. application Ser. No.11/972,459, entitled “Polypeptide Inhibitors of HSPB1 Kinase and UsesTherefor,” filed Jan. 10, 2008, and Ser. No. 12/188,109, entitled“Kinase Inhibitors and Uses Thereof,” filed Aug. 7, 2008, the contentsof each application are incorporated by reference in their entiretyherein).

11.1. Viral CPP Containing Proteins

The first proteins to be described as having transduction propertieswere of viral origin. These proteins still are the most commonlyaccepted models for CPP action. Among the cell-penetrating peptides, thearginine-rich cell-penetrating peptides, including but not limited toTAT peptide, have been the most widely studied (El-Sayed, A. et al.,AAPS J. 11, 13-22, 2009; Wender, P. et al., Adv. Drug Deliv. Rev. 60,452-472, 2008).

TAT (HIV-1 trans-activator gene product) is an 86-amino acidpolypeptide, which acts as a powerful transcription factor of theintegrated HIV-1 genome. TAT acts on the viral genome stimulating viralreplication in latently infected cells. The translocation properties ofthe TAT protein enable it to activate quiescent infected cells, and itmay be involved in priming of uninfected cells for subsequent infectionby regulating many cellular genes, including cytokines. The minimal CPPof TAT is the 9 amino acid protein sequence RKKRRQRRR (TAT49-57; SEQ IDNO: 20). Studies utilizing a longer fragment of TAT demonstratedsuccessful transduction of fusion proteins up to 120 kDa. The additionof multiple TAT-CPP as well as synthetic TAT derivatives has beendemonstrated to mediate membrane translocation. TAT CPP containingfusion proteins have been used as therapeutic moieties in experimentsinvolving cancer, transporting a death-protein into cells, and diseasemodels of neurodegenerative disorders.

VP22 is the HSV-1 tegument protein, a structural part of the HSV virion.VP22 is capable of receptor independent translocation and accumulates inthe nucleus. This property of VP22 classifies the protein as a CPPscontaining peptide. Fusion proteins comprising full length VP22 havebeen translocated efficiently across the plasma membrane.

11.2. Homeoproteins with Intercellular Translocation Properties

Homeoproteins are highly conserved, transactivating transcriptionfactors involved in morphological processes. They bind to DNA through aspecific sequence of 60 amino acids. The DNA-binding homeodomain is themost highly conserved sequence of the homeoprotein. Severalhomeoproteins have been described to exhibit CPP-like activity; they arecapable of efficient translocation across cell membranes in anenergy-independent and endocytosis-independent manner without cell typespecificity.

The Antennapedia protein (Antp) is a trans-activating factor capable oftranslocation across cell membranes; the minimal sequence capable oftranslocation is a 16 amino acid peptide corresponding to the thirdhelix of the protein's homeodomain (HD). The internalization of thishelix occurs at 4° C., suggesting that this process is not endocytosisdependent. Peptides up to 100 amino acids produced as fusion proteinswith AntpHD penetrate cell membranes.

Other homeodomains capable of translocation include Fushi tarazu (Ftz)and Engrailed (En) homeodomain. Many homeodomains share a highlyconserved third helix.

11.3. Human CPPs

Human CPPs may circumvent potential immunogenicity issues uponintroduction into a human patient. Peptides with CPPs sequences include:Hoxa-5, Hox-A4, Hox-B5, Hox-B6, Hox-B7, HOX-D3, GAX, MOX-2, and FtzCPP.These proteins all share the sequence found in AntpCPPs. Other CPPsinclude Islet-1, interleukin-1, tumor necrosis factor, and thehydrophobic sequence from Kaposi-fibroblast growth factor or FGF-4)signal peptide, which is capable of energy-, receptor-, andendocytosis-independent translocation. Unconfirmed CPPs include membersof the Fibroblast Growth Factor (FGF) family.

12. MK2 Inhibitors and Treatment of Fibrotic Diseases or Conditions

Mitogen-activated protein kinase activated protein kinase 2 (MAPKAPK2 orMK2), a serine/threonine kinase substrate downstream of p38MAPK, hasbeen implicated in many inflammatory diseases that are complicated byscarring and fibrosis (Lopes, L. et al., Biochem Biophys Res Commun.,382(3):535-9, 2009). These include, but are not limited to, cancer,intimal hyperplasia, organ fibrosis, abdominal adhesions, inflammatorybowel disease, and rheumatoid arthritis. In addition to idiopathicpulmonary fibrosis (IPF), other disorders that involve inflammation andfibrosis and impact the lung include acute lung injury (ALI), organtransplant rejection (with lung transplant also a later-stage treatmentfor IPF), organ failure secondary to sepsis, acute lung failure,auto-immune diseases such as scleroderma, and chronic pulmonaryobstructive disease (COPD).

The development of fibrosis is known to require inflammation,proliferation and recruitment of fibroblast that results in cells ofmyofibroblastic phenotype (Horowitz J. et al., Semin Respir Crit CareMed., 27(6):600-612, 2006). MK2 has been shown to control geneexpression at transcriptional and post-transcriptional levels (NeiningerA. et al., J Biol Chem. 2002; 277(5):3065-8, Thomas T. et al., JNeurochem., 105(5): 2039-52, 2008; Johansen C. et al., J Immunol.,176(3):1431-8, 2006; Rousseau S. et al., EMBO J. 21(23):6505-14, 2002)as well as cytoskeletal architecture (Lopes, L. et al., Biochem BiophysRes Commun., 382(3):535-9, 2009). In addition, it was shown thatactivated MK2 increases translation and stability of inflammatorycytokine mRNAs and causes actin reorganization; and that inhibition ofMK2 is associated with reduced inflammation (Ward, B. et al., J SurgRes., 169(1):e27-36, 2011) and myofibroblast differentiation (Lopes, L.et al., Biochem Biophys Res Commun., 382(3):535-9, 2009).

Together, these data suggest that inhibition of MK2 may providetherapeutic benefits to patients with fibrotic disorders or conditions,for example, idiopathic pulmonary fibrosis (IPF), acute lung injury(ALI), hepatic fibrosis, renal fibrosis, vascular fibrosis, andtransplant rejection. In this respect, the described invention offers anapproach to intervene in the process of inflammation and fibrosis usingcell-penetrating, peptide base inhibitors of MK2.

SUMMARY OF THE INVENTION

According to one aspect, the described invention provides a method forreducing progression of a fibrosis in a tissue selected from a livertissue, a kidney tissue or a vascular tissue, comprising: administeringto a subject in need thereof a pharmaceutical composition comprising atherapeutic amount of a polypeptide of the amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or a functional equivalent thereofmade from a fusion between a first polypeptide that is a cellpenetrating peptide (CPP) selected from the group consisting of apolypeptide of amino acid sequence WLRRIKAWLRRIKA (SEQ ID NO: 12),WLRRIKA (SEQ ID NO: 13), YGRKKRRQRRR (SEQ ID NO: 14), WLRRIKAWLRRI (SEQID NO: 15), FAKLAARLYR (SEQ ID NO: 16), KAFAKLAARLYR (SEQ ID NO: 17) andHRRIKAWLKKI (SEQ ID NO: 18), and a second polypeptide that is atherapeutic domain (TD) selected from the group consisting of apolypeptide of amino acid sequence KALARQLAVA (SEQ ID NO: 8), KALARQLGVA(SEQ ID NO: 9) and KALARQLGVAA (SEQ ID NO: 10), and a pharmaceuticallyacceptable carrier thereof, progression of the fibrosis beingcharacterized by one or more of aberrant fibroblast proliferation andextracellular matrix deposition producing remodeling in liver tissue,kidney tissue or vascular tissue, wherein the therapeutic amount of thepolypeptide is effective to reduce progression of the fibrosis, to treatremodeling of the tissue, or a combination thereof.

According to one embodiment, the fibrosis is further characterized by aninflammation in the tissue. According to another embodiment, theinflammation is an acute or a chronic inflammation. According to anotherembodiment, the inflammation is mediated by at least one cytokineselected from the group consisting of Tumor Necrosis Factor-alpha(TNF-α), Interleukin-6 (IL-6), and Interleukin-1β (IL-1β).

According to one embodiment, the aberrant fibroblast proliferation andextracellular matrix deposition in the tissue or the tissue ischaracterized by an aberrant activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 2 (MK2) in the tissue compared to theactivity of Mitogen-Activated Protein Kinase-Activated Protein Kinase 2(MK2) in the tissue of a normal healthy control subject, i.e., a subjecthaving no symptoms or other evidence of a liver, kidney or vascularfibrotic condition, disorder or disease.

According to one embodiment, the step of administering occurs orally,intratracheally, parenterally, intravenously, or intraperitoneally.

According to one embodiment, the pharmaceutical composition furthercomprises one or more additional therapeutic agent(s). According toanother embodiment, the additional therapeutic agent is ananti-infective agent. According to another embodiment, theanti-infective agent is an anti-viral agent. According to anotherembodiment, the antiviral agent is one or more of sofosbuvir (Sovaldi®),an HCV boosted protease inhibitor (ABT-450, AbbVie), a nonnucleosideNS5B inhibitor (dasabuvir, ABT-333, AbbVie), an NS5a inhibitor(ombitasvir, ABT-267, AbbVie), ABT-450/r (ABT-450 with ritonavir),ABT-450 co-formulated with ABT-267, or ABT-450 formulated withsofosbuvir, ribavirin, or combinations thereof.

According to one embodiment, the additional therapeutic agent is aglucocorticoid selected from the group consisting of prednisone,budesonide, mometasone furoate, fluticasone propionate, fluticasonefuroate, and a combination thereof.

According to one embodiment, the additional therapeutic agent is ananalgesic agent.

According to one embodiment, the additional therapeutic agent isselected from the group consisting of a purified bovine Type V collagen,an IL-13 receptor antagonist, a protein tyrosine kinase inhibitor, anendothelial receptor antagonist, a dual endothelin receptor antagonist,a prostacyclin analog, an anti-CTGF antibody, an endothelin receptorantagonist (A-selective), AB0024, a lysyl oxidase-like 2 (LOXL2)antibody, a c-Jun N-terminal kinase (JNK) inhibitor, pirfenidone,IFN-γ1b, anantibody against all three TGF-β isoforms, a pan-neutralizingIgG4 human antibody against all three TGF-β isoforms, a TGF-β activationinhibitor, a recombinant human Pentraxin-2 protein (rhPTX-2), abispecific IL-4/IL-13 antibody, an antibody targeting integrin αvβ6,N-acetylcysteine, sildenafil, a Tumor Necrosis Factor (TNF) antagonist(etanercept), and a combination thereof.

According to one embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence FAKLAARLYRKALARQLGVAA (SEQ ID NO: 3). According to anotherembodiment, the functional equivalent of the polypeptideYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acid sequenceKAFAKLAARLYRKALARQLGVAA (SEQ ID NO: 4). According to another embodiment,the functional equivalent of the polypeptide YARAAARQARAKALARQLGVAA (SEQID NO: 1) is of amino acid sequence YARAAARQARAKALARQLAVA (SEQ ID NO:5). According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence YARAAARQARAKALARQLGVA (SEQ ID NO: 6). According to anotherembodiment, the functional equivalent of the polypeptideYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acid sequenceHRRIKAWLKKIKALARQLGVAA (SEQ ID NO: 7). According to another embodiment,the functional equivalent of the polypeptide YARAAARQARAKALARQLGVAA (SEQID NO: 1) is of amino acid sequence YARAAARQARAKALNRQLAVAA (SEQ ID NO:26). According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence YARAARQARAKALNRQLAVA (SEQ ID NO: 27).

According to one embodiment, the second polypeptide of the functionalequivalent of the polypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) madefrom the fusion of the first polypeptide that is the cell penetratingpeptide (CPP) operatively linked to the second polypeptide that is thetherapeutic domain (TD) is a polypeptide whose sequence has asubstantial identity to amino acid sequence KALARQLGVAA (SEQ ID NO: 2).

According to one embodiment, the second polypeptide is a polypeptide ofamino acid sequence KALARQLAVA (SEQ ID NO: 8). According to anotherembodiment, the second polypeptide is a polypeptide of amino acidsequence KALARQLGVA (SEQ ID NO: 9). According to another embodiment, thesecond polypeptide is a polypeptide of amino acid sequence KALARQLGVAA(SEQ ID NO: 10). According to another embodiment, the second polypeptideis a polypeptide of amino acid sequence KALNRQLAVAA (SEQ ID NO: 28).According to another embodiment, the second polypeptide is a polypeptideof amino acid sequence KALNRQLAVA (SEQ ID NO: 29).

According to one embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is a fusion proteincomprising a first polypeptide operatively linked to a secondpolypeptide, wherein the first polypeptide is a cell penetrating peptidefunctionally equivalent to YARAAARQARA (SEQ ID NO: 11) selected from thegroup consisting of a polypeptide of amino acid sequence WLRRIKAWLRRIKA(SEQ ID NO: 12), WLRRIKA (SEQ ID NO: 13), YGRKKRRQRRR (SEQ ID NO: 14),WLRRIKAWLRRI (SEQ ID NO: 15), FAKLAARLYR (SEQ ID NO: 16), KAFAKLAARLYR(SEQ ID NO: 17) and HRRIKAWLKKI (SEQ ID NO: 18), and the secondpolypeptide is of amino acid sequence KALARQLGVAA (SEQ ID NO: 2).

According to one embodiment, the first polypeptide is a polypeptide ofamino acid sequence WLRRIKAWLRRIKA (SEQ ID NO: 12). According to anotherembodiment, the first polypeptide is a polypeptide of amino acidsequence WLRRIKA (SEQ ID NO: 13).

According to another embodiment, the first polypeptide is a polypeptideof amino acid sequence YGRKKRRQRRR (SEQ ID NO: 14). According to anotherembodiment, the first polypeptide is a polypeptide of amino acidsequence WLRRIKAWLRRI (SEQ ID NO: 15). According to another embodiment,the first polypeptide is a polypeptide of amino acid sequence FAKLAARLYR(SEQ ID NO: 16). According to another embodiment, the first polypeptideis a polypeptide of amino acid sequence KAFAKLAARLYR (SEQ ID NO: 17).According to another embodiment, the first polypeptide is a polypeptideof amino acid sequence HRRIKAWLKKI (SEQ ID NO: 18).

According to one embodiment, the carrier is selected from the groupconsisting of a controlled release carrier, a delayed release carrier, asustained release carrier, and a long-term release carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows delivery performance of neat spray-dried insulin.

FIG. 2 shows particle size distribution of spray-dried insulin, which isdetermined by Anderson Cascade Impaction (ACI).

FIG. 3 shows efficiency and flow rate comparison of MicroDose Dry PowderInhaler (DPI) vs. two marketed “passive” Dry Powder Inhalers (DPIs).

FIG. 4 shows flow-rate independence of spray-dried neat peptide.

FIG. 5 shows a representative micrograph of a spray-dried peptide (notinsulin).

FIG. 6 shows particle size distribution of a spray-dried peptide (notinsulin).

FIG. 7 shows particle size distribution of micronized/lactose blendcombination, which is determined by Next Generation Impactor (NGI)

FIG. 8 shows delivery performance of micronized a small molecule(long-acting muscarinic agents (LAMA)/lactose blend).

FIG. 9 shows immunohistochemical analysis of paraffin-embedded humanidiopathic pulmonary fibrosis IPF lungs, showing nuclear localization ofactivated MK2 (i.e., Phospho-Thr³³⁴-MAPKAPK2) at the fibroblastic focus.Normal lungs (left panel); IPF lung tissue biopsy section (right panel).Inset shows disruption of epithelial lining at the foci with cellsstaining positive (dark grey) for activated MK2. The abbreviations shownin FIG. 9 are as follows: NL (normal lung architecture with alveolarsacs); AW (air way); FF (fibroblastic foci from a lung tissue explantswith IPF)

FIG. 10 shows a schematic diagram for testing ability of a compound toinhibit the development of fibrosis in the bleomycin mouse model ofpulmonary fibrosis (Idiopathic Pulmonary Fibrosis (IPF) preventionmodel). Phosphate-buffered saline (PBS) or MMI-0100(YARAAARQARAKALARQLGVAA (SEQ ID NO: 1)) is administered daily, eithervia nebulization or intraperitoneally, starting at day 7 post bleomycindelivery when inflammation subsides and fibrotic mechanisms areactivated, until day 21 post bleomycin delivery when significantfibrosis is observed.

FIG. 11 shows that inhalation therapy and systemic administration ofMMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) protects againstbleomycin-induced lung fibrosis in mice. Upper panel: Hematoxylin andEosin (H&E) staining of representative mouse lung tissues at day 21.Lower panel: Masson's blue trichrome staining of the same fields revealextensive collagen deposition (arrows) with bleomycin injury.Abbreviations: AW: airway; NL: normal lung architecture; FF: fibroticfoci; V: vein.

FIG. 12 shows that MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)prevents significant collagen deposition due to bleomycin injury. Valuesrepresent means±SEM. n=5 animals per group. ‘*’ p<0.05; ‘**’ p<0.01;‘***’ p<0.001. Collagen Index=constant factor for collagen 7.5× hydroxyproline concentrations.

FIG. 13 shows that MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)prevents fibrosis due to bleomycin injury in a dose-dependent manner.Masson's blue trichrome staining of lung sections of bleomycin mice. (A)MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1); (B) MMI-0200(YARAAARQARAKALNRQLGVA; SEQ ID NO: 19).

FIG. 14 shows that systemically-administered MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) abrogates systemic T cellactivation due to bleomycin injury. Values represent mean±SEM. ‘p’ value<0.01. n=4 animals/group. The abbreviations shown in FIG. 14 are asfollows: (i) wild type mice treated with PBS (PBS); (ii) the bleomycinmice treated with PBS (BLEO); (iii) the bleomycin mice treated withnebulized MMI-0100 (YARAAARQARAKALARQLGVAA (SEQ ID NO: 1))(BLEO+MMI-0100 (NEB)); and (iv) the bleomycin mice treated withintraperitoneal MMI-0100 (YARAAARQARAKALARQLGVAA (SEQ ID NO: 1))(BLEO+MMI-0100 (IP)).

FIG. 15 shows a schematic diagram for testing ability of MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) to abrogate fibrosis progressionin the bleomycin model of idiopathic pulmonary fibrosis (IPF treatmentmodel). PBS or MMI-0100 (YARAAARQARAKALARQLGVAA (SEQ ID NO: 1)) isadministered via nebulization or intraperitoneally at the doses of 50μg/kg daily starting at day 14 post bleomycin delivery until day 28 postbleomycin delivery.

FIG. 16 shows that systemic (IP) or nebulized (NEB) administration ofMMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) amelioratesbleomycin-induced lung fibrosis in mice. Upper panel: Hematoxylin andEosin (H&E) staining; Lower panel: Masson's blue trichrome staining ofthe same fields. The abbreviations shown in FIG. 16 are as follows: PBS(wild type mice treated with PBS); BLEO (bleomycin mice treated withPBS); MMI-0100 (NEB) (bleomycin mice treated with nebulized MMI-0100(YARAAARQARAKALARQLGVAA (SEQ ID NO: 1)); MMI-0100 (IP) (bleomycin micetreated with intraperitoneal MMI-0100 (YARAAARQARAKALARQLGVAA (SEQ IDNO: 1)); NL (normal lung architecture with alveolar sacs); AW (air way);FF (fibroblastic foci from a lung tissue explants with IPF)

FIG. 17 shows that MMI-0100 (YARAAARQARAKALARQLGVAA (SEQ ID NO: 1))arrests significant collagen deposition due to bleomycin injury. Theabbreviations shown in FIG. 17 are as follows: PBS (wild type micetreated with PBS); BLEO (bleomycin mice treated with PBS); BLEO+MMI-0100(NEB) (bleomycin mice treated with nebulized MMI-0100(YARAAARQARAKALARQLGVAA (SEQ ID NO: 1)); BLEO+MMI-0100 (IP) (bleomycinmice treated with intraperitoneal MMI-0100 (YARAAARQARAKALARQLGVAA (SEQID NO: 1)). Values represent Means±SEM. n=5 animals per group. CollagenIndex=constant factor for collagen 7.5× hydroxyproline concentrations.

FIG. 18 shows a representative micrographs ofanti-phospho-Thr³³⁴-MAPKAPK2 (an activated form of MK2) staining of lungsections (at day 28 post bleomycin injury) from (i) wild type micetreated with PBS (PBS); (ii) bleomycin mice treated with PBS (BLEO);(iii) bleomycin mice treated with nebulized MMI-0100(YARAAARQARAKALARQLGVAA (SEQ ID NO: 1)) (BLEO+MMI-0100 (NEB)); and (iv)bleomycin mice treated with intraperitoneal MMI-0100(YARAAARQARAKALARQLGVAA (SEQ ID NO: 1)) (BLEO+MMI-0100 (IP)). C57-BL/6mice were subjected to bleomycin injury at day 0. At day 14, the micewere administered 50 μg/kg of MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ IDNO: 1) daily by intraperitoneal (IP) injection or nebulizer (NEB) untilday 28 post bleomycin injury. Original magnifications: 20×.

FIG. 19 shows key signaling molecules involved in TGF-β-mediatedinflammatory and fibrotic pathways.

FIG. 20 shows that, 24 hours after final administration, MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) downregulates the levels ofcirculating inflammatory cytokines in the bleomycin mouse model ofidiopathic pulmonary fibrosis (treatment model).

FIG. 21 shows that MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)inhibits myofibroblast alpha-smooth muscle actin (α-SMA) activation inthe idiopathic pulmonary fibrosis treatment model. C57-BL/6 mice weresubjected to bleomycin injury at day 0. At day 14 through day 28, micewere administered 50 μg/kg/day MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ IDNO: 1) by intraperitoneal (IP) injection or nebulizer (NEB).Formalin-fixed lung tissue sections were immunostained against α-SMA.Control staining was with biotinylated secondary IgG antibody.Streptavidin-conjugated horseradish peroxidase was used with3,3′-diaminobenzidene as substrate and nuclei was counterstained withhematoxylin. Original magnifications: 20×

FIG. 22 shows modulation of TGF-β-induced myofibroblast activation byMK2 peptide inhibitors in normal human fetal lung fibroblasts (IMR-90).IMR-90 cells were pre-treated with MMI-0100 (YARAAARQARAKALARQLGVAA; SEQID NO: 1) or MMI-0200 (YARAAARQARAKALNRQLGVA; SEQ ID NO: 19) at theindicated doses for 1 h and then cultured in the presence or absence ofTGF-β1 (2 ng/ml) for 48 h. Cell lysates were immunoblotted againstantibodies for α-SMA (a marker for myofibroblast activation) and GAPDH(loading control).

FIG. 23 shows modulation of TGF-β-mediated fibronectin expression inhuman fetal lung fibroblasts (IMR-90). IMR90 cells were pre-treated withMMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or MMI-0200(YARAAARQARAKALNRQLGVA; SEQ ID NO: 19) at the indicated doses for 1 h,and then cultured in the presence or absence of TGF-β1 (2 ng/ml) for 72h. Fibronection was measured as secreted fragments in the conditionedmedia. Equal amounts (14 μg) of total proteins from the conditionedmedia were loaded in each lane.

FIG. 24 shows key signaling molecules involved in the regulation ofmesenchymal stem cell migration by fibronectin throughα5β1-integrin-mediated activation of PDGFR-β (Veevers-Lowe J et al., JCell Sci, 124: 1288-1300, 2011).

FIG. 25 shows increases in the level of an MK2 kinase activated form inIPF patients. (A) quantitative analysis of phospho-Thr³³⁴ levels innormal and IPF tissues; (C) correlation between lung function and MK2activation.

FIG. 26 shows a schematic diagram of the pathogenetic mechanismunderlying renal fibrosis (Cho M H, Korean J. Pediatr. 2010; 53(7):735-740).

DETAILED DESCRIPTION OF THE INVENTION

The described invention provides a composition and method for reducingprogression of a fibrosis in a tissue selected from a liver tissue, akidney tissue or a vascular tissue in a subject in need of thereof, theprogression of which is characterized by one or more of aberrantfibroblast proliferation and extracellular matrix deposition producingtissue remodeling, the method comprising administering a therapeuticamount of a composition comprising a polypeptide having the amino acidsequence YARAAARQARAKALARQLGVAA (MMI-0100; SEQ ID NO: 1) or a functionalequivalent thereof, wherein the therapeutic amount of the polypeptide iseffective to reduce progression of the fibrosis, to treat remodeling ofthe tissue, or a combination thereof.

Glossary

The term “airway” as used herein refers to the passages through whichair enters and leaves the body. The pulmonary airway comprises thoseparts of the respiratory tract through which air passes duringbreathing.

The term “airway obstruction” as used herein refers to any abnormalreduction in airflow. Resistance to airflow can occur anywhere in theairway from the upper airway to the terminal bronchi.

The term “airway disease” as used herein refers to a disease thataffects the tubes (airways) that carry oxygen and other gases into andout of the lungs. Airway diseases include, but are not limited to,chronic obstructive pulmonary disease (COPD), including asthma,emphysema, and chronic bronchitis.

The term “lung tissue disease” as used herein refers to a disease thataffects the structure of the lung tissue, e.g., pulmonary interstitium.Scarring or inflammation of lung tissue makes the lungs unable to expandfully (“restrictive lung disease”). It also makes the lungs less capableof taking up oxygen (oxygenation) and releasing carbon dioxide. Examplesof lung tissue diseases include, but are not limited to, idiopathicpulmonary fibrosis (IPF), acute lung injury (ALI), a radiation-inducedfibrosis in the lung, and a fibrotic condition associated with lungtransplantation. Sarcoidosis is a disease in which swelling(inflammation) occurs in the lymph nodes, lungs, liver, eyes, skin, orother tissues.

The terms “lung interstitium” or “pulmonary interstitium” are usedinterchangeably herein to refer to an area located between the airspaceepithelium and pleural mesothelium in the lung. Fibers of the matrixproteins, collagen and elastin, are the major components of thepulmonary interstitium. The primary function of these fibers is to forma mechanical scaffold that maintains structural integrity duringventilation.

The term “accessible surface area” or “ASA” as used herein refers to asurface area of a biomolecule that is exposed to solvent. The term“solvent accessible surface” or “SAS” as used herein refers to apercentage of the surface area of a given residue that is accessible tothe solvent. It is calculated as a ratio between ASA of a residue in thethree dimensional structure and the maximum ASA of its extended peptideconfirmation

The terms “amino acid residue” or “amino acid” or “residue” are usedinterchangeably to refer to an amino acid that is incorporated into aprotein, a polypeptide, or a peptide, including, but not limited to, anaturally occurring amino acid and known analogs of natural amino acidsthat can function in a similar manner as naturally occurring aminoacids. The amino acids may be L- or D-amino acids. An amino acid may bereplaced by a synthetic amino acid, which is altered so as to increasethe half-life of the peptide, increase the potency of the peptide, orincrease the bioavailability of the peptide.

The single letter designation for amino acids is used predominatelyherein. As is well known by one of skill in the art, such single letterdesignations are as follows:

A is alanine; C is cysteine; D is aspartic acid; E is glutamic acid; Fis phenylalanine; G is glycine; H is histidine; I is isoleucine; K islysine; L is leucine; M is methionine; N is asparagine; P is proline; Qis glutamine; R is arginine; S is serine; T is threonine; V is valine; Wis tryptophan; and Y is tyrosine.

The following represents groups of amino acids that are conservativesubstitutions for one another: 1) Alanine (A), Serine (S), Threonine(T); 2) Aspartic Acid (D), Glutamic Acid (E); 3) Asparagine (N),Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine(L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W).

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. For example,reference to a “polypeptide” means one or more polypeptides.

The term “addition” as used herein refers to the insertion of one ormore bases, or of one or more amino acids, into a sequence.

The term “administer” as used herein refers to dispensing, supplying,applying, giving, apportioning or contributing. The terms“administering” or “administration” are used interchangeably and includein vivo administration, as well as administration directly to tissue exvivo. Generally, compositions may be administered systemically eitherorally, buccally, parenterally, topically, by inhalation or insufflation(i.e., through the mouth or through the nose), or rectally in dosageunit formulations containing the conventional nontoxic pharmaceuticallyacceptable carriers, adjuvants, and vehicles as desired, or may belocally administered by means such as, but not limited to, injection,implantation, grafting, topical application, or parenterally. Additionaladministration may be performed, for example, intravenously,pericardially, orally, via implant, transmucosally, transdermally,topically, intramuscularly, subcutaneously, intraperitoneally,intrathecally, intralymphatically, intralesionally, or epidurally.Administering can be performed, for example, once, a plurality of times,and/or over one or more extended periods.

The term “allergic reaction” as used herein refers to a hypersensitivereaction of the immune system. Allergic reactions occur to normallyharmless environmental substances known as allergens; these reactionsare acquired, predictable, and rapid. Allergic reaction is characterizedby excessive activation of certain white blood cells called mast cellsand basophils by a type of antibody known as IgE, resulting in anextreme inflammatory response. Common allergic reactions include eczema,hives, hay fever, asthma attacks, food allergies, and reactions to thevenom of stinging insects such as wasps and bees.

The term “α-smooth muscle actin” or “α-SMA” as used herein refers to anactin protein, alpha-actin-2 (ACTA2; also known as actin or aorticsmooth muscle actin) first isolated in vascular smooth muscle cells.Actins are highly conserved proteins expressed in all eukaryotic cells.Actin filaments form part of the cytoskeleton and play essential rolesin regulating cell shape and movement. Six distinct actin isotypes havebeen identified in mammalian cells. Each is encoded by a separated geneand is expressed in a developmentally regulated and tissue-specificmanner. Alpha and beta cytoplasmic actins are expressed in a widevariety of cells, whereas expression of alpha skeletal, alpha cardiac,alpha vascular, and gamma enteric actins are more restricted tospecialized muscle cell type. The gene for alpha-smooth muscle actin isone of a few genes whose expression is relatively restricted to vascularsmooth muscle cells, but it is now most commonly used as a marker ofmyofibroblast formation. Expression of alpha smooth muscle actin isregulated by hormones and cell proliferation, and is altered bypathological conditions, including oncogenic transformation andatherosclerosis.

The term “alveolus” or “alveoli” as used herein refers to an anatomicalstructure that has the form of a hollow cavity. Found in the lung, thepulmonary alveoli are spherical outcroppings of the respiratory sites ofgas exchange with the blood. The alveoli contain some collagen andelastic fibers. Elastic fibers allow the alveoli to stretch as they fillwith air when breathing in. They then spring back during breathing outin order to expel the carbon dioxide-rich air.

The term “biomarker” (or “biosignature”) as used herein refers to apeptide, a protein, a nucleic acid, an antibody, a gene, a metabolite,or any other substance used as an indicator of a biologic state. It is acharacteristic that is measured objectively and evaluated as a cellularor molecular indicator of normal biologic processes, pathogenicprocesses, or pharmacologic responses to a therapeutic intervention.

The term “bleomycin” as used herein refers to a glycopeptide antibioticproduced by the bacterium Streptomyces verticillus. It works by inducingDNA strand breaks and inhibiting incorporation of thymidine into DNAstrand. The most serious complication of bleomycin is pulmonary fibrosisand impaired lung function.

The term “bronchoalveolar lavage” or “BAL” as used herein refers to amedical procedure in which a bronchoscope is passed through the mouth ornose into the lungs and fluid is squirted into a small part of the lungand then recollected for examination. BAL typically is performed todiagnose lung disease. BAL commonly is used to diagnose infections inpeople with immune system problems, pneumonia in people on ventilators,some types of lung cancer, and scarring of the lung (interstitial lungdisease). BAL is the most common manner to sample the components of theepithelial lining fluid (ELF) and to determine the protein compositionof the pulmonary airways, and is often used in immunological research asa means of sampling cells or pathogen levels in the lung.

The terms “carrier” and “pharmaceutical carrier” as used herein refer toa pharmaceutically acceptable inert agent or vehicle for delivering oneor more active agents to a subject, and often is referred to as“excipient.” The (pharmaceutical) carrier must be of sufficiently highpurity and of sufficiently low toxicity to render it suitable foradministration to the subject being treated. The (pharmaceutical)carrier further should maintain the stability and bioavailability of anactive agent, e.g., a polypeptide of the described invention. The(pharmaceutical) carrier can be liquid or solid and is selected, withthe planned manner of administration in mind, to provide for the desiredbulk, consistency, etc., when combined with an active agent and othercomponents of a given composition. The (pharmaceutical) carrier may be,without limitation, a binding agent (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.), a filler(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates, calciumhydrogen phosphate, etc.), a lubricant (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.), a disintegrant (e.g., starch, sodiumstarch glycolate, etc.), or a wetting agent (e.g., sodium laurylsulphate, etc.). Other suitable (pharmaceutical) carriers for thecompositions of the described invention include, but are not limited to,water, salt solutions, alcohols, polyethylene glycols, gelatins,amyloses, magnesium stearates, talcs, silicic acids, viscous paraffins,hydroxymethylcelluloses, polyvinylpyrrolidones and the like.Compositions that are for parenteral administration of a polypeptide ofthe described invention may include (pharmaceutical) carriers such assterile aqueous solutions, non-aqueous solutions in common solvents suchas alcohols, or solutions of the polypeptide in a liquid oil base.

The term “collagen” as used herein refers to a group of naturallyoccurring proteins found in the flesh and in connective tissues ofmammals. It is the main component of connective tissue, and is the mostabundant protein in mammals, making up about 25% to 35% of thewhole-body protein content. Collagen, in the form of elongated fibrils,is mostly found in fibrous tissues, such as tendon, ligament, and skin,and is also abundant in cornea, cartilage, bone, blood vessels, the gut,and intervertebral disc. So far, 29 types of collagen have beenidentified and over 90% of the collagen in the body is of type I (skin,tendon, vascular, ligature, organs, bone), type II (cartilage), type III(reticulate (main component of reticular fibers), and type IV (whichforms the bases of cell base membrane).

The term “condition” as used herein refers to a variety of health statesand is meant to include disorders or diseases caused by any underlyingmechanism, disorder, or injury.

The term “cytokine,” which refers to small soluble protein substancessecreted by cells that have a variety of effects on other cells, isgenerically used to refer to many signaling molecules including, withoutlimitation, lymphokines, interleukins, and chemokines. Cytokines mediatemany important physiological functions including growth, development,wound healing, and the immune response. They act by binding to theircell-specific receptors located in the cell membrane that allows adistinct signal transduction cascade to start in the cell, whicheventually will lead to biochemical and phenotypic changes in targetcells. Generally, cytokines act locally, although some have been foundto have systemic immunomodulatory effects, with pleiotropic autocrine,paracrine, and endocrine effects similar to hormones. They include typeI cytokines, which encompass many of the interleukins, as well asseveral hematopoietic growth factors; type II cytokines, including theinterferons and interleukin-10; tumor necrosis factor (“TNF”)-relatedmolecules, including TNF-α and lymphotoxin; immunoglobulin super-familymembers, including interleukin 1 (“IL-1”); and the chemokines, a familyof molecules that play a critical role in a wide variety of immune andinflammatory functions. The same cytokine can have different effects ona cell depending on the state of the cell. Cytokines often regulate theexpression of, and trigger cascades of, other cytokines.

The terms “disease” or “disorder” as used herein refer to an impairmentof health or a condition of abnormal functioning, regardless of cause(whether heritable, environmental, dietary, infectious, due to trauma,or otherwise). Disorders may include, for example, but are not limitedto, inflammatory and fibrotic diseases, fibrosis, acute lung injury,radiation-induced fibrosis, transplant rejection, chronic obstructivepulmonary disease (COPD), endotoxic shock, localized inflammatorydisease, atherosclerotic cardiovascular disease, Alzheimer's disease,oncological diseases, neural ischemia, connective tissue and systemicautoimmune diseases, rheumatoid arthritis, Crohn's disease, inflammatorybowel disease, systemic lupus erythematosus (SLE), Sjögren's syndrome,scleroderma, vasculitis, intimal hyperplasia, stenosis, restenosis,atherosclerosis, smooth muscle cell tumors and metastasis, smooth musclespasm, angina, Prinzmetal's angina, ischemia, stroke, bradycardia,hypertension, cardiac hypertrophy, renal failure, stroke, pulmonaryhypertension, asthma, toxemia of pregnancy, pre-term labor,pre-eclampsia, eclampsia, Raynaud's disease or phenomenon,hemolytic-uremia, anal fissure, achalasia, impotence, migraine, ischemicmuscle injury associated with smooth muscle spasm, vasculopathy,bradyarrythmia, congestive heart failure, stunned myocardium, pulmonaryhypertension, diastolic dysfunction, gliosis (proliferation ofastrocytes, and may include deposition of extracellular matrix (ECM)deposition in damaged areas of the central nervous system), chronicobstructive pulmonary disease (i.e., respiratory tract diseasescharacterized by airflow obstruction or limitation; includes, but is notlimited to, chronic bronchitis, emphysema, and chronic asthma),osteopenia, endothelial dysfunction, inflammation, degenerativearthritis, anklyosing spondylitis, Guillain-Barré disease, infectiousdisease, sepsis, endotoxemic shock, psoriasis, radiation enteritis,cirrhosis, interstitial fibrosis, pulmonary fibrosis (includingidiopathic pulmonary fibrosis), colitis, appendicitis, gastritis,laryngitis, meningitis, pancreatitis, otitis, reperfusion injury,traumatic brain injury, spinal cord injury, peripheral neuropathy,multiple sclerosis, allergy, cardiometabolic diseases, obesity, type IIdiabetes mellitus, type I diabetes mellitis, and NASH/cirrhosis.

The term “domain” as used herein refers to a region of a protein with acharacteristic tertiary structure and function and to any of thethree-dimensional subunits of a protein that together makes up itstertiary structure formed by folding its linear peptide chain.

The term “therapeutic domain” (also referred to as “TD”) as used hereinrefers to a peptide, peptide segment or variant, or derivative thereof,with substantial identity to peptide KALARQLGVAA (SEQ ID NO: 2), orsegment thereof. Therapeutic domains by themselves generally are notcapable of penetrating the plasma membrane of mammalian cells. Onceinside the cell, therapeutic domains can inhibit the kinase activity ofa specific group of kinases.

The term “cell penetrating peptide” (also referred to as “CPP,” “proteintransduction domain,” “PTD”, “Trojan peptide”, “membrane translocatingsequence”, and “cell permeable protein”) as used herein refers to aclass of peptides generally capable of penetrating the plasma membraneof mammalian cells. It also refers to a peptide, peptide segment, orvariant or derivative thereof, with substantial identity to peptideYARAAARQARA (SEQ ID NO: 11), or a functional segment thereof, and to apeptide, peptide segment, or variant or derivative thereof, which isfunctionally equivalent to SEQ ID NO: 11. CPPs generally are 10-16 aminoacids in length and are capable of transporting compounds of many typesand molecular weights across mammalian cells. Such compounds include,but are not limited to, effector molecules, such as proteins, DNA,conjugated peptides, oligonucleotides, and small particles such asliposomes. CPPs chemically linked or fused to other proteins (“fusionproteins”) still are able to penetrate the plasma membrane and entercells.

The term “extracellular matrix” as used herein refers to a scaffold in acell's external environment with which the cell interacts via specificcell surface receptors. The extracellular matrix is composed of aninterlocking mesh of fibrous proteins and glycosaminoglycans (GAGs).Examples of fibrous proteins found in the extracellular matrix include,without limitation, collagen, elastin, fibronectin, and laminin.Examples of GAGs found in the extracellular matrix include, withoutlimitation, proteoglycans (e.g., heparin sulfate), chondroitin sulfate,keratin sulfate, and non-proteoglycan polysaccharide (e.g., hyaluronicacid). The term “proteoglycan” refers to a group of glycoproteins thatcontain a core protein to which is attached one or moreglycosaminoglycans. The extracellular matrix serves many functions,including, but not limited to, providing support and anchorage forcells, segregating one tissue from another tissue, and regulatingintracellular communication.

The terms “functional equivalent” or “functionally equivalent” are usedinterchangeably herein to refer to substances, molecules,polynucleotides, proteins, peptides, or polypeptides having similar oridentical effects or use. A polypeptide functionally equivalent topolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1), for example, may havea biologic activity, e.g., an inhibitory activity, kinetic parameters,salt inhibition, a cofactor-dependent activity, and/or a functional unitsize that is substantially similar or identical to the expressedpolypeptide of SEQ ID NO: 1.

Examples of polypeptides functionally equivalent toYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) include, but are not limited to, apolypeptide of amino acid sequence FAKLAARLYRKALARQLGVAA (SEQ ID NO: 3),a polypeptide of amino acid sequence KAFAKLAARLYRKALARQLGVAA (SEQ ID NO:4), a polypeptide of amino acid sequence YARAAARQARAKALARQLAVA (SEQ IDNO: 5), a polypeptide of amino acid sequence YARAAARQARAKALARQLGVA (SEQID NO: 6), a polypeptide of amino acid sequence YARAAARQARAKALNRQLAVAA(SEQ ID NO: 26), a polypeptide of amino acid sequenceYARAARQARAKALNRQLAVA (SEQ ID NO: 27) and a polypeptide of amino acidsequence HRRIKAWLKKIKALARQLGVAA (SEQ ID NO: 7).

The MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) peptide of aminoacid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) described in thepresent invention comprises a fusion protein in which a cell penetratingpeptide (CPP; YARAAARQARA; SEQ ID NO: 11) is operatively linked to atherapeutic domain (KALARQLGVAA; SEQ ID NO: 2) in order to enhancetherapeutic efficacy.

Examples of polypeptides functionally equivalent to the therapeuticdomain (TD; KALARQLGVAA; SEQ ID NO: 2) of the polypeptideYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) include, but are not limited to, apolypeptide of amino acid sequence KALARQLAVA (SEQ ID NO: 8), apolypeptide of amino acid sequence KALARQLGVA (SEQ ID NO: 9), apolypeptide of amino acid sequence KALARQLGVAA (SEQ ID NO: 10), apolypeptide of amino acid sequence KALNRQLAVAA (SEQ ID NO: 28), and apolypeptide of amino acid sequence KALNRQLAVA (SEQ ID NO: 29).

Examples of polypeptides functionally equivalent to the cell penetratingpeptide (CPP; YARAAARQARA; SEQ ID NO: 11) of the polypeptideYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) include, but are not limited to, apolypeptide of amino acid sequence WLRRIKAWLRRIKA (SEQ ID NO: 12), apolypeptide of amino acid sequence WLRRIKA (SEQ ID NO: 13), apolypeptide of amino acid sequence YGRKKRRQRRR (SEQ ID NO: 14), apolypeptide of amino acid sequence WLRRIKAWLRRI (SEQ ID NO: 15), apolypeptide of amino acid sequence FAKLAARLYR (SEQ ID NO: 16), apolypeptide of amino acid sequence KAFAKLAARLYR (SEQ ID NO: 17), and apolypeptide of amino acid sequence HRRIKAWLKKI (SEQ ID NO: 18).

The term “endogenous” as used herein refers to growing or originatingfrom within, or derived internally.

The term “endothelium” as used herein refers to a thin layer of cellsthat lines the interior surface of blood vessels, forming an interfacebetween circulating blood in the lumen and the rest of the vessel wall.Endothelial cells will line the entire circulatory system, from theheart to the smallest capillary. These cells reduce turbulence of theflow of blood allowing the fluid to be pumped farther.

The term “eosinophils” or “eosinophil granulocytes” as used hereinrefers to white blood cells responsible for combating multicellularparasites and certain infections in vertebrates. They are granulocytesthat develop during hematopoiesis in the bone marrow before migratinginto blood. Along with mast cells, they also control mechanismsassociated with allergy and asthma. Following activation, eosinophilsexert diverse functions, including (1) production of cationic granuleproteins and their release by degranulation, (2) production of reactiveoxygen species, such as, superoxide, peroxide, and hypobromite(hypobromous acid, which is preferentially produced by eosinophilperoxidase), (3) production of lipid mediators, such as, eicosanoidsfrom leukotriene and prostaglandin families, (4) production of growthfactors, such as transforming growth factor (TGF-β), vascularendothelial growth factor (VEGF), and platelet-derived growth factor(PDGF), and (5) production of cytokines such as IL-1, IL-2, IL-4, IL-5,IL-6, IL-8, IL-13, and TNF-α.

The term “epithelium” as used herein refers to a tissue composed ofcells that line the cavities and surfaces of structures throughout thebody. The basal surface of the epithelium faces underlying connectivetissue, and the two layers are separated by a basement membrane.

The term “extravasation” as used herein refers to the movement of bloodcell components from the capillaries to the tissues surrounding them(diapedesis). In the case of malignant cancer metastasis, it refers tocancer cells exiting the capillaries and entering organs.

The term “exudation” as used herein refers to a process by which a fluidfrom the circulatory system passes through the walls of the bloodvessels into lesions or areas of inflammation. Blood exudates containsome or all plasma proteins, white blood cells, platelets and red bloodcells.

The term “fibrin” as used herein refers to a fibrous protein involved inthe clotting of blood. It is a fibrillar protein that is polymerized toform a “mesh” that forms a hemostatic plug or clot (in conjunction withplatelets) over a wound site. Fibrin is involved in signal transduction,blood coagulation, platelet activation, and protein polymerization.

The term “fibroblast” as used herein refers to a connective tissue cellthat makes and secretes the extracellular matrix proteins, including,but not limited to, collagen. Fibroblasts, the most common cell typefound in connective tissues, play an important role in healing wounds.Like other cells of connective tissue, fibroblasts are derived fromprimitive mesenchyme (a type of loose connective tissue derived from allthree germ layers and located in the embryos). In certain situationsepithelial cells can give rise to fibroblasts, a process calledepithelial-mesenchymal transition. Fibroblasts and fibrocytes are twostates of the same cells, the former being the activated state, thelatter the less active state, concerned with maintenance and tissuemetabolism, with both terms occasionally used interchangeably.

The term “free radical” as used herein refers to a highly reactive andusually short-lived molecular fragment with one or more unpairedelectrons. Free radicals are highly chemically reactive molecules.Because a free radical needs to extract a second electron from aneighboring molecule to pair its single electron, it often reacts withother molecules, which initiates the formation of many more free radicalspecies in a self-propagating chain reaction. This ability to beself-propagating makes free radicals highly toxic to living organisms.Oxidative injury may lead to widespread biochemical damage within thecell. The molecular mechanisms responsible for this damage are complex.For example, free radicals may damage intracellular macromolecules, suchas nucleic acids (e.g., DNA and RNA), proteins, and lipids. Free radicaldamage to cellular proteins may lead to loss of enzymatic function andcell death. Free radical damage to DNA may cause problems in replicationor transcription, leading to cell death or uncontrolled cell growth.Free radical damage to cell membrane lipids may cause the damagedmembranes to lose their ability to transport oxygen, nutrients or waterto cells.

The term “myofibroblasts” as used herein refers to fibroblasts in woundareas that have some characteristics of smooth muscle, such ascontractile properties and fibers, and are believed to produce,temporarily, type III collagen. Although there are many possible ways ofmyofibroblast development, myofibroblasts are cells that are in betweenfibroblasts and smooth muscle cells in their differentiation. In manyorgans like liver, lung, and kidney they are primarily involved infibrosis. In wound tissue, they are implicated in wound strengthening(by extracellular collagen fiber deposition) and then wound contraction(by intracellular contraction and concomitant alignment of the collagenfibers by integrin mediated pulling o to the collagen bundles).

The term “fibronectin” as used herein refers to a high-molecular weight(˜440 kDa) extracellular matrix glycoprotein that binds tomembrane-spanning cell-surface matrix receptor proteins (“integrins”)and to extracellular matrix components such as collagen, fibrin andheparan sulfate proteoglycans (e.g. syndecans). Fibronectin exists as adimer, consisting of two nearly identical monomers linked by a pair ofdisulfide bonds. There are multiple isoforms of fibronectin. Plasmafibronectin is soluble and circulates in the blood and other bodyfluids, where it is thought to enhance blood clotting, wound healing andphagocytosis. The other isoforms assemble on the surface of cells andare deposited in the extracellular matrix as highly insolublefibronectin fibrils. The fibronectin fibrils that form on or near thesurface of fibroblasts usually are aligned with adjacent intracellularactin stress fibers, which promote the assembly of secreted fibronectinmolecules into fibrils and influence fibril orientation. Fibronectinplays a major role in cell adhesion, cell growth, cell migration andcell differentiation, and it is important for processes such as woundhealing and embryonic development.

The term “fibrosis” as used herein refers to the formation ordevelopment of excess fibrous connective tissue in an organ or tissue asa result of injury or inflammation of a part, or of interference withits blood supply. It may be a consequence of the normal healing responseleading to a scar, an abnormal, reactive process, or without known orunderstood causation.

The term “inhalation” as used herein refers to the act of drawing in amedicated vapor with the breath.

The term “insufflation” as used herein refers to the act of deliveringair, a gas, or a powder under pressure to a cavity or chamber of thebody. For example, nasal insufflation relates to the act of deliveringair, a gas, or a powder under pressure through the nose.

The term “inhalation delivery device” as used herein refers to amachine/apparatus or component that produces small droplets or anaerosol from a liquid or dry powder aerosol formulation and is used foradministration through the mouth in order to achieve pulmonaryadministration of a drug, e.g., in solution, powder, and the like.Examples of inhalation delivery device include, but are not limited to,a nebulizer, a metered-dose inhaler, and a dry powder inhaler (DPI).

The term “nebulizer” as used herein refers to a device used toadminister liquid medication in the form of a mist inhaled into thelungs.

The term “metered-dose inhaler”, “MDI”, or “puffer” as used hereinrefers to a pressurized, hand-held device that uses propellants todeliver a specific amount of medicine (“metered dose”) to the lungs of apatient. The term “propellant” as used herein refers to a material thatis used to expel a substance usually by gas pressure through aconvergent, divergent nozzle. The pressure may be from a compressed gas,or a gas produced by a chemical reaction. The exhaust material may be agas, liquid, plasma, or, before the chemical reaction, a solid, liquidor gel. Propellants used in pressurized metered dose inhalers areliquefied gases, traditionally chlorofluorocarbons (CFCs) andincreasingly hydrofluoroalkanes (HFAs). Suitable propellants include,for example, a chlorofluorocarbon (CFC), such as trichlorofluoromethane(also referred to as propellant 11), dichlorodifluoromethane (alsoreferred to as propellant 12), and1,2-dichloro-1,1,2,2-tetrafluoroethane (also referred to as propellant114), a hydrochlorofluorocarbon, a hydrofluorocarbon (HFC), such as1,1,1,2-tetrafluoroethane (also referred to as propellant 134a,HFC-134a, or HFA-134a) and 1,1,1,2,3,3,3-heptafluoropropane (alsoreferred to as propellant 227, HFC-227, or HFA-227), carbon dioxide,dimethyl ether, butane, propane, or mixtures thereof. In otherembodiments, the propellant includes a chlorofluorocarbon, ahydrochlorofluorocarbon, a hydrofluorocarbon, or mixtures thereof. Inother embodiments, a hydrofluorocarbon is used as the propellant. Inother embodiments, HFC-227 and/or HFC-134a are used as the propellant.

The term “dry powder inhaler” or “DPI” as used herein refers to a devicesimilar to a metered-dose inhaler, but where the drug is in powder form.The patient exhales out a full breath, places the lips around themouthpiece, and then quickly breathes in the powder. Dry powder inhalersdo not require the timing and coordination that are necessary with MDIs.

The term “particles” as used herein refers to refers to an extremelysmall constituent (e.g., nanoparticles, microparticles, or in someinstances larger) in or on which is contained the composition asdescribed herein.

The terms “pulmonary fibrosis”, “idiopathic pulmonary fibrosis”, and“cryptogenic fibrosing alveolitis” as used herein refer to a majorcomponent of interstitial lung disease characterized by abnormalfibroblast proliferation and deposition of extracellular matrix proteinsthat remodel the normal pulmonary tissue structure and compromise itsfunction. The hallmark lesions of idiopathic pulmonary fibrosis are thefibroblast foci. These sites feature vigorous replication of mesenchymalcells and exuberant deposition of fresh extracellular matrix.

The terms “fibrotic loci” or “fibrotic foci” as used hereininterchangeably refer to a specific location in a tissue formed ordeveloped by excessive fibrous tissue.

The term “fusion protein” as used herein refers to a protein orpolypeptide constructed by combining multiple protein domains orpolypeptides for the purpose of creating a single polypeptide or proteinwith functional properties derived from each of the original proteins orpolypeptides. Creation of a fusion protein may be accomplished byoperatively ligating or linking two different nucleotides sequences thatencode each protein domain or polypeptide via recombinant DNAtechnology, thereby creating a new polynucleotide sequences that codesfor the desired fusion protein. Alternatively, a fusion protein maybecreated by chemically joining the desired protein domains.

The term “idiopathic” as used herein means arising spontaneously or froman obscure or unknown cause.

The term “inflammation” as used herein refers to the physiologic processby which vascularized tissues respond to injury. See, e.g., FUNDAMENTALIMMUNOLOGY, 4th Ed., William E. Paul, ed. Lippincott-Raven Publishers,Philadelphia (1999) at 1051-1053, incorporated herein by reference.During the inflammatory process, cells involved in detoxification andrepair are mobilized to the compromised site by inflammatory mediators.Inflammation is often characterized by a strong infiltration ofleukocytes at the site of inflammation, particularly neutrophils(polymorphonuclear cells). These cells promote tissue damage byreleasing toxic substances at the vascular wall or in uninjured tissue.Traditionally, inflammation has been divided into acute and chronicresponses.

The term “acute inflammation” as used herein refers to the rapid,short-lived (minutes to days), relatively uniform response to acuteinjury characterized by accumulations of fluid, plasma proteins, andneutrophilic leukocytes. Examples of injurious agents that cause acuteinflammation include, but are not limited to, pathogens (e.g., bacteria,viruses, parasites), foreign bodies from exogenous (e.g. asbestos) orendogenous (e.g., urate crystals, immune complexes), sources, andphysical (e.g., burns) or chemical (e.g., caustics) agents.

The term “chronic inflammation” as used herein refers to inflammationthat is of longer duration and which has a vague and indefinitetermination. Chronic inflammation takes over when acute inflammationpersists, either through incomplete clearance of the initialinflammatory agent (e.g., cigarette smoking) or as a result of multipleacute events occurring in the same location. Chronic inflammation, whichincludes the influx of lymphocytes and macrophages and fibroblastgrowth, may result in tissue scarring at sites of prolonged or repeatedinflammatory activity.

The term “inflammatory mediators” as used herein refers to the molecularmediators of the inflammatory and immune processes. These soluble,diffusible molecules act both locally at the site of tissue damage andinfection and at more distant sites. Some inflammatory mediators areactivated by the inflammatory process, while others are synthesizedand/or released from cellular sources in response to acute inflammationor by other soluble inflammatory mediators; still others exhibitanti-inflammatory properties. Examples of inflammatory mediators of theinflammatory response include, but are not limited to, plasma proteases,complement, kinins, clotting and fibrinolytic proteins, lipid mediators,prostaglandins, leukotrienes, platelet-activating factor (PAF),peptides, hormones (including steroid hormones such as glucocorticoids),and amines, including, but not limited to, histamine, serotonin, andneuropeptides, and proinflammatory cytokines, including, but not limitedto, interleukin-1-beta (IL-1β), interleukin-4 (IL-4), interleukin-6(IL-6), interleukin-8 (IL-8), tumor necrosis factor-alpha (TNF-α),interferon-gamma (IF-γ), interleukin-12 (IL-12), and interleukin-17(IL-17).

Among the pro-inflammatory mediators, IL-1, IL-6, and TNF-α are known toactivate hepatocytes in an acute phase response to synthesizeacute-phase proteins that activate complement. Complement is a system ofplasma proteins that interact with pathogens to mark them fordestruction by phagocytes. Complement proteins can be activated directlyby pathogens or indirectly by pathogen-bound antibody, leading to acascade of reactions that occurs on the surface of pathogens andgenerates active components with various effector functions. IL-1, IL-6,and TNF-α also activate bone marrow endothelium to mobilize neutrophils,and function as endogenous pyrogens, raising body temperature, whichhelps eliminating infections from the body. A major effect of thecytokines is to act on the hypothalamus, altering the body's temperatureregulation, and on muscle and fat cells, stimulating the catabolism ofthe muscle and fat cells to elevate body temperature. At elevatedtemperatures, bacterial and viral replications are decreased, while theadaptive immune system operates more efficiently.

The term “tumor necrosis factor” as used herein refers to a cytokinemade by white blood cells in response to an antigen or infection, whichinduce necrosis (death) of tumor cells and possesses a wide range ofpro-inflammatory actions. Tumor necrosis factor also is amultifunctional cytokine with effects on lipid metabolism, coagulation,insulin resistance, and the function of endothelial cells lining bloodvessels.

The term “interleukin (IL)” as used herein refers to a cytokine from aclass of homologously related proteins that were first observed to besecreted by, and acting on, leukocytes. It has since been found thatinterleukins are produced by a wide variety of body cells. Interleukinsregulate cell growth, differentiation, and motility, and stimulatesimmune responses, such as inflammation. Examples of interleukinsinclude, interleukin-1 (IL-1), interleukin-1β (IL-1β), interleukin-6(IL-6), interleukin-8 (IL-8), interleukin-12 (IL-12), and interleukin-17(IL-17).

The terms “inhibiting”, “inhibit” or “inhibition” are used herein torefer to reducing the amount or rate of a process, to stopping theprocess entirely, or to decreasing, limiting, or blocking the action orfunction thereof. Inhibition may include a reduction or decrease of theamount, rate, action function, or process of a substance by at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 45%, at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95%, at least 98%, or at least 99%.

The term “inhibitor” as used herein refers to a second molecule thatbinds to a first molecule thereby decreasing the first molecule'sactivity. Enzyme inhibitors are molecules that bind to enzymes therebydecreasing enzyme activity. The binding of an inhibitor may stop asubstrate from entering the active site of the enzyme and/or hinder theenzyme from catalyzing its reaction. Inhibitor binding is eitherreversible or irreversible. Irreversible inhibitors usually react withthe enzyme and change it chemically, for example, by modifying key aminoacid residues needed for enzymatic activity. In contrast, reversibleinhibitors bind non-covalently and produce different types of inhibitiondepending on whether these inhibitors bind the enzyme, theenzyme-substrate complex, or both. Enzyme inhibitors often are evaluatedby their specificity and potency.

The term “injury” as used herein refers to damage or harm to a structureor function of the body caused by an outside agent or force, which maybe physical or chemical.

The term “isolated” is used herein to refer to material, such as, butnot limited to, a nucleic acid, peptide, polypeptide, or protein, whichis: (1) substantially or essentially free from components that normallyaccompany or interact with it as found in its naturally occurringenvironment. The terms “substantially free” or “essentially free” areused herein to refer to considerably or significantly free of, or morethan about 95% free of, or more than about 99% free of such components.The isolated material optionally comprises material not found with thematerial in its natural environment; or (2) if the material is in itsnatural environment, the material has been synthetically (non-naturally)altered by deliberate human intervention to a composition and/or placedat a location in the cell (e.g., genome or subcellular organelle) notnative to a material found in that environment. The alteration to yieldthe synthetic material may be performed on the material within, orremoved, from its natural state. For example, a naturally occurringnucleic acid becomes an isolated nucleic acid if it is altered, or if itis transcribed from DNA that has been altered, by means of humanintervention performed within the cell from which it originates. See,for example, Compounds and Methods for Site Directed Mutagenesis inEukaryotic Cells, Kmiec, U.S. Pat. No. 5,565,350; In Vivo HomologousSequence Targeting in Eukaryotic Cells; Zarling et al., PCT/US93/03868,each incorporated herein by reference in its entirety. Likewise, anaturally occurring nucleic acid (for example, a promoter) becomesisolated if it is introduced by non-naturally occurring means to a locusof the genome not native to that nucleic acid. Nucleic acids that are“isolated” as defined herein also are referred to as “heterologous”nucleic acids.

The term “kinase” as used herein refers to a type of enzyme thattransfers phosphate groups from high-energy donor molecules to specifictarget molecules or substrates. High-energy donor groups may include,but are not limited, to ATP.

The term “leukocyte” or “white blood cell (WBC)” as used herein refersto a type of immune cell. Most leukocytes are made in the bone marrowand are found in the blood and lymph tissue. Leukocytes help the bodyfight infections and other diseases. Granulocytes, monocytes, andlymphocytes are leukocytes.

The term “lymphocytes” as used herein refers to a small white blood cell(leukocyte) that plays a large role in defending the body againstdisease. There are two main types of lymphocytes: B cells and T cells.The B cells make antibodies that attack bacteria and toxins while the Tcells themselves attack body cells when they have been taken over byviruses or have become cancerous. Lymphocytes secrete products(lymphokines) that modulate the functional activities of many othertypes of cells and are often present at sites of chronic inflammation.

The term “macrophage” as used herein refers to a type of white bloodcell that surrounds and kills microorganisms, removes dead cells, andstimulates the action of other immune system cells. After digesting apathogen, a macrophage presents an antigen (a molecule, most often aprotein found on the surface of the pathogen, used by the immune systemfor identification) of the pathogen to the corresponding helper T cell.The presentation is done by integrating it into the cell membrane anddisplaying it attached to an MHC class II molecule, indicating to otherwhite blood cells that the macrophage is not a pathogen, despite havingantigens on its surface. Eventually, the antigen presentation results inthe production of antibodies that attach to the antigens of pathogens,making them easier for macrophages to adhere to with their cell membraneand phagocytose.

The term “mesenchymal cell” or “mesenchyme” as used herein refers to acell derived from all three germ layers, which can develop intoconnective tissue, bone, cartilage, the lymphatic system, and thecirculatory system.

The term “MK2 kinase” or “MK2” as used herein refers tomitogen-activated protein kinase-activated protein kinase 2 (alsoreferred to as “MAPKAPK2”, “MAPKAP-K2”, “MK2”), which is a member of theserine/threonine (Ser/Thr) protein kinase family.

The term “mass median aerodynamic diameter” or “MMAD” as used hereinrefers to median of the distribution of airborne particle mass withrespect to the aerodynamic diameter. MMADs are usually accompanied bythe geometric standard deviation (g or sigma g), which characterizes thevariability of the particle size distribution.

The term “modulate” as used herein means to regulate, alter, adapt, oradjust to a certain measure or proportion.

The term “monocyte” as used herein refers to a type of immune cell thatis made in the bone marrow and travels through the blood to tissues inthe body where it becomes a macrophage. A monocyte is a type of whiteblood cell and a type of phagocyte.

The term “neutrophils” or “polymorphonuclear neutrophils (PMNs)” as usedherein refers to the most abundant type of white blood cells in mammals,which form an essential part of the innate immune system. They form partof the polymorphonuclear cell family (PMNs) together with basophils andeosinophils. Neutrophils are normally found in the blood stream. Duringthe beginning (acute) phase of inflammation, particularly as a result ofbacterial infection and some cancers, neutrophils are one of thefirst-responders of inflammatory cells to migrate toward the site ofinflammation. They migrate through the blood vessels, then throughinterstitial tissue, following chemical signals such as interleukin-8(IL-8) and C5a in a process called chemotaxis, the directed motion of amotile cell or part along a chemical concentration gradient towardenvironmental conditions it deems attractive and/or away fromsurroundings it finds repellent.

The term “normal healthy control subject” as used herein refers to asubject having no symptoms or other clinical evidence of airway or lungtissue disease.

The term “nucleic acid” is used herein to refer to a deoxyribonucleotideor ribonucleotide polymer in either single- or double-stranded form, andunless otherwise limited, encompasses known analogues having theessential nature of natural nucleotides in that they hybridize tosingle-stranded nucleic acids in a manner similar to naturally occurringnucleotides (e.g., peptide nucleic acids).

The term “nucleotide” is used herein to refer to a chemical compoundthat consists of a heterocyclic base, a sugar, and one or more phosphategroups. In the most common nucleotides, the base is a derivative ofpurine or pyrimidine, and the sugar is the pentose deoxyribose orribose. Nucleotides are the monomers of nucleic acids, with three ormore bonding together in order to form a nucleic acid. Nucleotides arethe structural units of RNA, DNA, and several cofactors, including, butnot limited to, CoA, FAD, DMN, NAD, and NADP. Purines include adenine(A), and guanine (G); pyrimidines include cytosine (C), thymine (T), anduracil (U).

The following terms are used herein to describe the sequencerelationships between two or more nucleic acids or polynucleotides: (a)“reference sequence”, (b) “comparison window”, (c) “sequence identity”,(d) “percentage of sequence identity”, and (e) “substantial identity.”

(a) The term “reference sequence” refers to a sequence used as a basisfor sequence comparison. A reference sequence may be a subset or theentirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) The term “comparison window” refers to a contiguous and specifiedsegment of a polynucleotide sequence, wherein the polynucleotidesequence may be compared to a reference sequence and wherein the portionof the polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two sequences. Generally, the comparison window is at least 20contiguous nucleotides in length, and optionally can be at least 30contiguous nucleotides in length, at least 40 contiguous nucleotides inlength, at least 50 contiguous nucleotides in length, at least 100contiguous nucleotides in length, or longer. Those of skill in the artunderstand that to avoid a high similarity to a reference sequence dueto inclusion of gaps in the polynucleotide sequence, a gap penaltytypically is introduced and is subtracted from the number of matches.

Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math.2:482 (1981); by the homology alignment algorithm of Needleman andWunsch, J. Mol. Biol. 48:443 (1970); by the search for similarity methodof Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444 (1988); bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 575 ScienceDr., Madison, Wis., USA; the CLUSTAL program is well described byHiggins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS5:151-153 (1989); Corpet, et al., Nucleic Acids Research 16:10881-90(1988); Huang, et al., Computer Applications in the Biosciences,8:155-65 (1992), and Pearson, et al., Methods in Molecular Biology,24:307-331 (1994). The BLAST family of programs, which can be used fordatabase similarity searches, includes: BLASTN for nucleotide querysequences against nucleotide database sequences; BLASTX for nucleotidequery sequences against protein database sequences; BLASTP for proteinquery sequences against protein database sequences; TBLASTN for proteinquery sequences against nucleotide database sequences; and TBLASTX fornucleotide query sequences against nucleotide database sequences. See,Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995).

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using the BLAST 2.0 suite of programsusing default parameters. Altschul et al., Nucleic Acids Res.25:3389-3402 (1997). Software for performing BLAST analyses is publiclyavailable, e.g., through the National Center forBiotechnology-Information. This algorithm involves first identifyinghigh scoring sequence pairs (HSPs) by identifying short words of lengthW in the query sequence, which either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighborhood wordscore threshold (Altschul et al., supra). These initial neighborhoodword hits act as seeds for initiating searches to find longer HSPscontaining them. The word hits then are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Cumulative scores are calculated using, for nucleotidesequences, the parameters M (reward score for a pair of matchingresidues; always >0) and N (penalty score for mismatching residues;always <0). For amino acid sequences, a scoring matrix is used tocalculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a word length (W) of 11, anexpectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison ofboth strands. For amino acid sequences, the BLASTP program uses asdefaults a word length (W) of 3, an expectation (E) of 10, and theBLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. BLAST searches assume thatproteins may be modeled as random sequences. However, many real proteinscomprise regions of nonrandom sequences which may be homopolymerictracts, short-period repeats, or regions enriched in one or more aminoacids. Such low-complexity regions may be aligned between unrelatedproteins even though other regions of the protein are entirelydissimilar. A number of low-complexity filter programs may be employedto reduce such low-complexity alignments. For example, the SEG (Wootenand Federhen, Comput. Chem., 17:149-163 (1993)) and XNU (Claverie andStates, Comput. Chem., 17:191-201 (1993)) low-complexity filters may beemployed alone or in combination.

(c) The term “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences is used herein to refer to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions that are not identical often differ by conservativeamino acid substitutions, i.e., where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g. charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well-known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller, Computer Applic. Biol. Sci., 4:11-17(1988) e.g., as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA).

(d) The term “percentage of sequence identity” is used herein mean thevalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%sequence identity, at least 80% sequence identity, at least 90% sequenceidentity and at least 95% sequence identity, compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill will recognize that these values may beadjusted appropriately to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning and thelike. Substantial identity of amino acid sequences for these purposesnormally means sequence identity of at least 60%, or at least 70%, atleast 80%, at least 90%, or at least 95%. Another indication thatnucleotide sequences are substantially identical is if two moleculeshybridize to each other under stringent conditions. However, nucleicacids that do not hybridize to each other under stringent conditions arestill substantially identical if the polypeptides that they encode aresubstantially identical. This may occur, e.g., when a copy of a nucleicacid is created using the maximum codon degeneracy permitted by thegenetic code. One indication that two nucleic acid sequences aresubstantially identical is that the polypeptide that the first nucleicacid encodes is immunologically cross reactive with the polypeptideencoded by the second nucleic acid.

The phrase “operatively linked” as used herein refers to a linkage inwhich two or more protein domains or polypeptides are ligated orcombined via recombinant DNA technology or chemical reaction such thateach protein domain or polypeptide of the resulting fusion proteinretains its original function. For example, SEQ ID NO: 1 is constructedby operatively linking a cell penetrating peptide (SEQ ID NO: 11) with atherapeutic domain (SEQ ID NO: 2), thereby creating a fusion peptidethat possesses both the cell penetrating function of SEQ ID NO: 11 andthe kinase inhibitor function of SEQ ID NO: 2.

The term “oxidative stress” as used herein refers to a disturbance inthe balance between the production of reactive oxygen species (freeradicals) and antioxidant defenses.

The term “parenchyma” as used herein refers to an animal tissue thatconstitutes the essential part of an organ as contrasted with connectivetissue or blood vessels. The term “parenchymal” means pertaining to theparenchyma of an organ.

The term “parenteral” as used herein refers to introduction into thebody by way of an injection (i.e., administration by injection),including, for example, subcutaneously (i.e., an injection beneath theskin), intramuscularly (i.e., an injection into a muscle), intravenously(i.e., an injection into a vein), intrathecally (i.e., an injection intothe space around the spinal cord or under the arachnoid membrane of thebrain), intrasternal injection or infusion techniques, and includingintraperitoneal injection or infusion into the body cavity (e.g.peritoneum). A parenterally administered composition is delivered usinga needle, e.g., a surgical needle, or other corporal access device. Theterm “surgical needle” as used herein, refers to any access deviceadapted for delivery of fluid (i.e., capable of flow) compositions intoa selected anatomical structure. Injectable preparations, such assterile injectable aqueous or oleaginous suspensions, may be formulatedaccording to the known art using suitable dispersing or wetting agentsand suspending agents.

The term “particulate” as used herein refers to fine particles of solidor liquid matter suspended in a gas or liquid.

As used herein the term “pharmaceutically acceptable carrier” refers toany substantially non-toxic carrier conventionally useable foradministration of pharmaceuticals in which the isolated polypeptide ofthe present invention will remain stable and bioavailable. Thepharmaceutically acceptable carrier must be of sufficiently high purityand of sufficiently low toxicity to render it suitable foradministration to the mammal being treated. It further should maintainthe stability and bioavailability of an active agent. Thepharmaceutically acceptable carrier can be liquid or solid and isselected, with the planned manner of administration in mind, to providefor the desired bulk, consistency, etc., when combined with an activeagent and other components of a given composition.

The term “pharmaceutically acceptable salt” means those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like and are commensurate with areasonable benefit/risk ratio.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids.

The terms “polypeptide” and “protein” also are used herein in theirbroadest sense to refer to a sequence of subunit amino acids, amino acidanalogs, or peptidomimetics. The subunits are linked by peptide bonds,except where noted. The polypeptides described herein may be chemicallysynthesized or recombinantly expressed. Polypeptides of the describedinvention also can be synthesized chemically. Synthetic polypeptides,prepared using the well known techniques of solid phase, liquid phase,or peptide condensation techniques, or any combination thereof, caninclude natural and unnatural amino acids. Amino acids used for peptidesynthesis may be standard Boc (N-α-amino protectedN-α-t-butyloxycarbonyl) amino acid resin with the standard deprotecting,neutralization, coupling and wash protocols of the original solid phaseprocedure of Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154), or thebase-labile N-α-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) aminoacids first described by Carpino and Han (1972, J. Org. Chem.37:3403-3409). Both Fmoc and Boc N-α-amino protected amino acids can beobtained from Sigma, Cambridge Research Biochemical, or other chemicalcompanies familiar to those skilled in the art. In addition, thepolypeptides can be synthesized with other N-α-protecting groups thatare familiar to those skilled in this art. Solid phase peptide synthesismay be accomplished by techniques familiar to those in the art andprovided, for example, in Stewart and Young, 1984, Solid PhaseSynthesis, Second Edition, Pierce Chemical Co., Rockford, Ill.; Fieldsand Noble, 1990, Int. J. Pept. Protein Res. 35:161-214, or usingautomated synthesizers. The polypeptides of the invention may compriseD-amino acids (which are resistant to L-amino acid-specific proteases invivo), a combination of D- and L-amino acids, and various “designer”amino acids (e.g., β-methyl amino acids, C-α-methyl amino acids, andN-α-methyl amino acids, etc.) to convey special properties. Syntheticamino acids include ornithine for lysine, and norleucine for leucine orisoleucine. In addition, the polypeptides can have peptidomimetic bonds,such as ester bonds, to prepare peptides with novel properties. Forexample, a peptide may be generated that incorporates a reduced peptidebond, i.e., R1-CH₂—NH—R2, where R1 and R2 are amino acid residues orsequences. A reduced peptide bond may be introduced as a dipeptidesubunit. Such a polypeptide would be resistant to protease activity, andwould possess an extended half-live in vivo. Accordingly, these termsalso apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, the protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids.

The terms “polypeptide”, “peptide” and “protein” also are inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation, and ADP-ribosylation. It will be appreciated, as is wellknown and as noted above, that polypeptides may not be entirely linear.For instance, polypeptides may be branched as a result ofubiquitination, and they may be circular, with or without branching,generally as a result of posttranslational events, including naturalprocessing event and events brought about by human manipulation which donot occur naturally. Circular, branched and branched circularpolypeptides may be synthesized by non-translation natural process andby entirely synthetic methods, as well. In some embodiments, the peptideis of any length or size.

The term “proenzyme” or “zymogen” as used herein refers to an inactiveenzyme precursor. A zymogen requires a biochemical change (such as ahydrolysis reaction revealing the active site, or changing theconfiguration to reveal the active site) for it to become an activeenzyme. The biochemical change usually occurs in a lysosome where aspecific part of the precursor enzyme is cleaved in order to activateit. The amino acid chain that is released upon activation is called theactivation peptide.

The term “proliferation” as used herein refers to expansion of apopulation of cells by the continuous division of single cells intoidentical daughter cells.

The term “pulmonary interstitium” as used herein refers to the tissueand space around the air sacs of the lungs.

The term “pulmonary alveolus” as used herein refers to an anatomicalstructure that has the form of a hollow cavity. The alveoli are locatedin the respiratory zone of the lungs, at the distal termination of thealveolar ducts and atria, forming the termination point of therespiratory tract. The pulmonary alveoli are spherical outcroppings ofthe respiratory sites of gas exchange with the blood and only found inthe mammalian lungs. The alveolar membrane is the gas-exchange surface.The blood brings carbon dioxide from the rest of the body for releaseinto the alveoli, and the oxygen in the alveoli is taken up by the bloodin the alveolar blood vessels, to be transported to all the cells in thebody. The alveoli contain some collagen and elastic fibers. The elasticfibers allow the alveoli to stretch as they fill with air when breathingin. They then spring back during breathing out in order to expel thecarbon dioxide-rich air. There are three major alveolar cell types inthe alveolar wall, (1) sequamous alveolar cells that form the structureof an alveolar wall, (2) great alveolar cells that secrete pulmonarysurfactant to lower the surface tension of water and allows the membraneto separate, thereby increasing the capability to exchange gasses, (3)macrophages that destroy foreign pathogens, such as bacteria.

The term “reactive oxygen species” (“ROS”), such as free radicals andperoxides, as used herein refers to a class of molecules that arederived from the metabolism of oxygen and exist inherently in allaerobic organisms. The term “oxygen radicals” as used herein refers toany oxygen species that carries an unpaired electron (except freeoxygen). The transfer of electrons to oxygen also may lead to theproduction of toxic free radical species. The best documented of theseis the superoxide radical. Oxygen radicals, such as the hydroxyl radical(OH—) and the superoxide ion (O2-) are very powerful oxidizing agentsthat cause structural damage to proteins, lipids and nucleic acids. Thefree radical superoxide anion, a product of normal cellular metabolism,is produced mainly in mitochondria because of incomplete reduction ofoxygen. The superoxide radical, although unreactive compared with manyother radicals, may be converted by biological systems into other morereactive species, such as peroxyl (ROO—), alkoxyl (RO—) and hydroxyl(OH—) radicals.

The phrase “remodeling in a tissue”, “tissue remodeling” and the like asused herein refers to the dynamic process that occurs during fetal oradult life and involves a modification of the original organization andfunction of the tissue.

The term “similar” is used interchangeably with the terms analogous,comparable, or resembling, meaning having traits or characteristics incommon.

The term “solution” as used herein refers to a homogeneous mixture oftwo or more substances. It is frequently, though not necessarily, aliquid. In a solution, the molecules of the solute (or dissolvedsubstance) are uniformly distributed among those of the solvent.

The terms “soluble” and “solubility” refer to the property of beingsusceptible to being dissolved in a specified fluid (solvent). The term“insoluble” refers to the property of a material that has minimal orlimited solubility in a specified solvent. In a solution, the moleculesof the solute (or dissolved substance) are uniformly distributed amongthose of the solvent.

The term “stress fiber” as used herein refers to high order structuresin cells consisting of actin filaments, crosslinking proteins (proteinsthat bind two or more filaments together), and myosin II motors. Actinis a globular protein (˜43 kDa), which polymerizes and forms into anordered filament structure which has two protofilaments wrapping aroundeach other, to form a single “actin filament” also known as a“microfilament.” The myosin motors in the stress fibers move, slidingactin filaments past one another, so the fiber can contract. In orderfor contraction to generate forces, the fibers must be anchored tosomething. Stress fibers can anchor to the cell membrane, and frequentlythe sites where this anchoring occurs are also connected to structuresoutside the cell (the matrix or some other substrate). These connectionsites are called focal adhesions. Many proteins are required for properfocal adhesion production and maintenance. Contraction against thesefixed external substrates is what allows the force generated by myosinmotors and filament growth and rearrangement to move and reshape thecell.

The term “suspension” as used herein refers to a dispersion (mixture) inwhich a finely-divided species is combined with another species, withthe former being so finely divided and mixed that it doesn't rapidlysettle out. In everyday life, the most common suspensions are those ofsolids in liquid.

The terms “subject” or “individual” or “patient” are usedinterchangeably to refer to a member of an animal species of mammalianorigin, including but not limited to, a mouse, a rat, a cat, a goat,sheep, horse, hamster, ferret, platypus, pig, a dog, a guinea pig, arabbit and a primate, such as, for example, a monkey, ape, or human.

The phrase “subject in need of such treatment” as used herein refers toa patient who suffers from a disease, disorder, condition, orpathological process. In some embodiments, the term “subject in need ofsuch treatment” also is used to refer to a patient who (i) will beadministered at least one polypeptide of the invention; (ii) isreceiving at least one polypeptide of the invention; or (iii) hasreceived at least one polypeptide of the invention, unless the contextand usage of the phrase indicates otherwise.

The term “substitution” is used herein to refer to a situation in whicha base or bases are exchanged for another base or bases in a DNAsequence. Substitutions may be synonymous substitutions or nonsynonymoussubstitutions. As used herein, “synonymous substitutions” refer tosubstitutions of one base for another in an exon of a gene coding for aprotein, such that the amino acid sequence produced is not modified. Theterm “nonsynonymous substitutions” as used herein refer to substitutionsof one base for another in an exon of a gene coding for a protein, suchthat the amino acid sequence produced is modified.

The terms “therapeutic amount,” an “amount effective,” or“pharmaceutically effective amount” of an active agent are usedinterchangeably to refer to an amount that is sufficient to provide theintended benefit of treatment. For example, the “therapeutic amount” ofa kinase inhibiting composition of the described invention includes, butis not limited to, an amount sufficient: (1) to remove, or decrease thesize of, at least one fibrotic locus or (2) to reduce the rate ofextracellular matrix, including collagen and fibronectin, deposition inthe interstitia in the lungs of a pulmonary fibrosis patient. The termalso encompasses an amount sufficient to suppress or alleviate at leastone symptom of a pulmonary fibrosis patient, wherein the symptomincludes, but is not limited to, oxygen saturation, dyspnea (difficultybreathing), nonproductive cough (meaning a sudden, noisy expulsion ofair from the lungs that may be caused by irritation or inflammation anddoes not remove sputum from the respiratory tract), clubbing (adisfigurement of the fingers into a bulbous appearance), and crackles(crackling sound in lungs during inhalation, occasionally refered to asrales or crepitations).

An effective amount of an active agent that can be employed according tothe described invention generally ranges from generally about 0.001mg/kg body weight to about 10 g/kg body weight. However, dosage levelsare based on a variety of factors, including the type of injury, theage, weight, sex, medical condition of the patient, the severity of thecondition, the route and frequency of administration, and the particularactive agent employed. Thus the dosage regimen may vary widely, but canbe determined routinely by a physician using standard methods.

The term “treat” or “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a disease, conditionor disorder, substantially ameliorating clinical or esthetical symptomsof a condition, substantially preventing the appearance of clinical oresthetical symptoms of a disease, condition, or disorder, and protectingfrom harmful or annoying symptoms. Treating further refers toaccomplishing one or more of the following: (a) reducing the severity ofthe disorder; (b) limiting development of symptoms characteristic of thedisorder(s) being treated; (c) limiting worsening of symptomscharacteristic of the disorder(s) being treated; (d) limiting recurrenceof the disorder(s) in patients that have previously had the disorder(s);and (e) limiting recurrence of symptoms in patients that were previouslyasymptomatic for the disorder(s).

The terms “variants”, “mutants”, and “derivatives” are used herein torefer to nucleotide or polypeptide sequences with substantial identityto a reference nucleotide or polypeptide sequence. The differences inthe sequences may be the result of changes, either naturally or bydesign, in sequence or structure. Natural changes may arise during thecourse of normal replication or duplication in nature of the particularnucleic acid sequence. Designed changes may be specifically designed andintroduced into the sequence for specific purposes. Such specificchanges may be made in vitro using a variety of mutagenesis techniques.Such sequence variants generated specifically may be referred to as“mutants” or “derivatives” of the original sequence.

A skilled artisan likewise can produce polypeptide variants ofpolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) having single ormultiple amino acid substitutions, deletions, additions or replacements,but functionally equivalent to SEQ ID NO: 1. These variants may includeinter alia: (a) variants in which one or more amino acid residues aresubstituted with conservative or non-conservative amino acids; (b)variants in which one or more amino acids are added; (c) variants inwhich at least one amino acid includes a substituent group; (d) variantsin which amino acid residues from one species are substituted for thecorresponding residue in another species, either at conserved ornon-conserved positions; and (d) variants in which a target protein isfused with another peptide or polypeptide such as a fusion partner, aprotein tag or other chemical moiety, that may confer useful propertiesto the target protein, for example, an epitope for an antibody. Thetechniques for obtaining such variants, including, but not limited to,genetic (suppressions, deletions, mutations, etc.), chemical, andenzymatic techniques, are known to the skilled artisan. As used herein,the term “mutation” refers to a change of the DNA sequence within a geneor chromosome of an organism resulting in the creation of a newcharacter or trait not found in the parental type, or the process bywhich such a change occurs in a chromosome, either through an alterationin the nucleotide sequence of the DNA coding for a gene or through achange in the physical arrangement of a chromosome. Three mechanisms ofmutation include substitution (exchange of one base pair for another),addition (the insertion of one or more bases into a sequence), anddeletion (loss of one or more base pairs).

The term “vehicle” as used herein refers to a substance that facilitatesthe use of a drug or other material that is mixed with it.

The term “wound healing” or “wound repair” as used herein refersgenerally to the body's natural process of repairing tissue aftertrauma. When an individual is wounded, a set of complex biochemicalevents takes place to repair the damage including, hemostasis,inflammation, proliferation, and remodeling.

I. Compositions: Therapeutic Peptides for Preventing or TreatingDiseases Characterized by Aberrant Fibroblast Proliferation and CollagenDeposition

According to one aspect, the described invention provides apharmaceutical composition for use in the treatment of a disease,condition, or process characterized by aberrant fibroblast proliferationand extracellular matrix deposition in a tissue of a subject,

wherein the pharmaceutical composition comprises a therapeutic amount ofa polypeptide of the amino acid sequence YARAAARQARAKALARQLGVAA (SEQ IDNO: 1) or a functional equivalent thereof, and a pharmaceuticallyacceptable carrier thereof, and

wherein the therapeutic amount is effective to reduce the fibroblastproliferation and extracellular matrix deposition in the tissue of thesubject.

According to one embodiment, the disease or the condition is Acute LungInjury (ALI) or acute respiratory distress syndrome (ARDS).

According to another embodiment, the disease or the condition isradiation-induced fibrosis.

According to another embodiment, the disease or the condition istransplant rejection.

According to another embodiment, the tissue is a lung tissue.

According to another embodiment, the disease or the condition is aninterstitial lung disease.

According to another embodiment, wherein the disease or the condition ispulmonary fibrosis.

According to another embodiment, wherein the pulmonary fibrosis isidiopathic pulmonary fibrosis.

According to another embodiment, the pulmonary fibrosis results fromadministration of bleomycin.

According to another embodiment, the pulmonary fibrosis results from anallergic reaction, inhalation of environmental particulates, smoking, abacterial infection, a viral infection, mechanical damage to a lung ofthe subject, lung transplantation rejection, an autoimmune disorder, agenetic disorder, or a combination thereof.

According to another embodiment, the tissue is a liver tissue.

According to another embodiment, the disease or condition is liverfibrosis.

According to another embodiment, the tissue is a kidney tissue.

According to another embodiment, the disease or condition is renalfibrosis.

According to another embodiment, the tissue is a vascular tissue.

According to another embodiment, the disease or condition is vascularfibrosis.

According to another embodiment, the disease or the condition is furthercharacterized by an inflammation in the tissue.

According to another embodiment, the inflammation is an acute or achronic inflammation.

According to another embodiment, the inflammation is mediated by atleast one cytokine selected from the group consisting of Tumor NecrosisFactor-alpha (TNF-α), Interleukin-6 (IL-6), and Interleukin-1β (IL-1β).

According to another embodiment, the pulmonary fibrosis is characterizedby at least one pathology selected from the group consisting of anaberrant deposition of an extracellular matrix protein in a pulmonaryinterstitium, an aberrant promotion of fibroblast proliferation in thelung, an aberrant induction of myofibroblast differentiation in thelung, and an aberrant promotion of attachment of myofibroblasts to anextracellular matrix compared to a normal healthy control subject.

According to another embodiment, the aberrant fibroblast proliferationand extracellular matrix deposition in the tissue is characterized by anaberrant activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2) in the tissue compared to the activity ofMitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2) in thetissue of a normal healthy control subject.

According to another embodiment, the aberrant fibroblast proliferationand extracellular matrix deposition in the tissue is evidenced by anaberrant amount or distribution of activated (phosphorylated)Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2) in thetissue compared to the amount or distribution of activatedMitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2) in thetissue of a normal healthy control subject.

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of a kinase selected from the group listed in Table 1herein.

According to another embodiment, this inhibition may, for example, beeffective to reduce fibroblast proliferation, extracellular matrixdeposition, or a combination thereof in the tissue of the subject.

According to another embodiment, this inhibition may, for example, beeffective to reduce at least one pathology selected from the groupconsisting of an aberrant deposition of an extracellular matrix proteinin a pulmonary interstitium, an aberrant promotion of fibroblastproliferation in the lung, an aberrant induction of myofibroblastdifferentiation, and an aberrant promotion of attachment ofmyofibroblasts to an extracellular matrix, compared to a normal healthycontrol subject.

According to some embodiments, inhibitory profiles of MMI inhibitors invivo depend on dosages, routes of administration, and cell typesresponding to the inhibitors.

According to another embodiment, the pharmaceutical composition inhibitsat least 50% of the kinase activity of the kinase. According to anotherembodiment, the pharmaceutical composition inhibits at least 65% of thekinase activity of the kinase. According to another embodiment, thepharmaceutical composition inhibits at least 75% of the kinase activityof that kinase. According to another embodiment, the pharmaceuticalcomposition inhibits at least 80% of the kinase activity of that kinase.According to another embodiment, the pharmaceutical composition inhibitsat least 85% of the kinase activity of that kinase. According to anotherembodiment, the pharmaceutical composition inhibits at least 90% of thekinase activity of that kinase. According to another embodiment, thepharmaceutical composition inhibits at least 95% of the kinase activityof that kinase.

According to some embodiments, the pharmaceutical composition inhibits akinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2 kinase). According to some other embodiments, thepharmaceutical composition inhibits at least 50% of the kinase activityof MK2 kinase. According to some other embodiments, the pharmaceuticalcomposition inhibits at least 65% of the kinase activity of MK2 kinase.According to another embodiment, the pharmaceutical composition inhibitsat least 75% of the kinase activity of MK2 kinase. According to anotherembodiment, the pharmaceutical composition inhibits at least 80% of thekinase activity of MK2 kinase. According to another embodiment, thepharmaceutical composition inhibits at least 85% of the kinase activityof MK2 kinase. According to another embodiment, the pharmaceuticalcomposition inhibits at least 90% of the kinase activity of MK2 kinase.According to another embodiment, the pharmaceutical composition inhibitsat least 95% of the kinase activity of MK2 kinase.

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 3 (MK3 kinase). According to another embodiment, thepharmaceutical composition inhibits at least 50% of the kinase activityof MK3 kinase. According to another embodiment, the pharmaceuticalcomposition inhibits at least 65% of the kinase activity of MK3 kinase.According to another embodiment, the pharmaceutical composition inhibitsat least 70% of the kinase activity of MK3 kinase. According to anotherembodiment, the pharmaceutical composition inhibits at least 75% of thekinase activity of MK3 kinase. According to another embodiment, thepharmaceutical composition inhibits at least 80% of the kinase activityof MK3 kinase. According to another embodiment, the pharmaceuticalcomposition inhibits at least 85% of the kinase activity of MK3 kinase.According to another embodiment, the pharmaceutical composition inhibitsat least 90% of the kinase activity of MK3 kinase. According to anotherembodiment, the pharmaceutical composition inhibits at least 95% of thekinase activity of MK3 kinase.

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of calcium/calmodulin-dependent protein kinase I(CaMKI). According to another embodiment, the pharmaceutical compositionfurther inhibits at least 50% of the kinase activity ofCa²⁺/calmodulin-dependent protein kinase I (CaMKI). According to anotherembodiment, the pharmaceutical composition further inhibits at least 65%of the kinase activity of Ca²⁺/calmodulin-dependent protein kinase I(CaMKI). According to another embodiment, the pharmaceutical compositionfurther inhibits at least 70% of the kinase activity ofCa²⁺/calmodulin-dependent protein kinase I (CaMKI). According to anotherembodiment, the pharmaceutical composition further inhibits at least 75%of the kinase activity of Ca²⁺/calmodulin-dependent protein kinase I(CaMKI). According to another embodiment, the pharmaceutical compositionfurther inhibits at least 80% of the kinase activity ofCa²⁺/calmodulin-dependent protein kinase I (CaMKI). According to anotherembodiment, the pharmaceutical composition further inhibits at least 85%of the kinase activity of Ca²⁺/calmodulin-dependent protein kinase I(CaMKI). According to another embodiment, the pharmaceutical compositionfurther inhibits at least 90% of the kinase activity ofCa²⁺/calmodulin-dependent protein kinase I (CaMKI). According to anotherembodiment, the pharmaceutical composition further inhibits at least 95%of the kinase activity of Ca²⁺/calmodulin-dependent protein kinase I(CaMKI).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of BDNF/NT-3 growth factors receptor (TrkB). Accordingto another embodiment, the pharmaceutical further inhibits at least 50%of the kinase activity of BDNF/NT-3 growth factors receptor (TrkB).According to another embodiment, the pharmaceutical further inhibits atleast 65% of the kinase activity of BDNF/NT-3 growth factors receptor(TrkB). According to another embodiment, the pharmaceutical furtherinhibits at least 70% of the kinase activity of BDNF/NT-3 growth factorsreceptor (TrkB). According to another embodiment, the pharmaceuticalfurther inhibits at least 75% of the kinase activity of BDNF/NT-3 growthfactors receptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2) and a kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 3 (MK3).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2) and a kinase activity of calcium/calmodulin-dependentprotein kinase I (CaMKI).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2) and a kinase activity of BDNF/NT-3 growth factorsreceptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2), a kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 3 (MK3), a kinase activity ofcalcium/calmodulin-dependent protein kinase I (CaMKI), and a kinaseactivity of BDNF/NT-3 growth factors receptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2), a kinase activity of calcium/calmodulin-dependentprotein kinase I (CaMKI), and a kinase activity of BDNF/NT-3 growthfactors receptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 2 (MK2).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 3 (MK3).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of calcium/calmodulin-dependentprotein kinase I (CaMKI).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of BDNF/NT-3 growth factors receptor(TrkB).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 2 (MK2) and at least 65% of the kinaseactivity of Mitogen-Activated Protein Kinase-Activated Protein Kinase 3(MK3).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 2 (MK2) and at least 65% of the kinaseactivity of calcium/calmodulin-dependent protein kinase I (CaMKI).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 2 (MK2) and at least 65% of the kinaseactivity of BDNF/NT-3 growth factors receptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 2 (MK2), at least 65% of the kinaseactivity of Mitogen-Activated Protein Kinase-Activated Protein Kinase 3(MK3), at least 65% of the kinase activity ofcalcium/calmodulin-dependent protein kinase I (CaMKI), and at least 65%of the kinase activity of BDNF/NT-3 growth factors receptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsthe kinase activity of at least one kinase selected from the group ofMK2, MK3, CaMKI, TrkB, without substantially inhibiting the activity ofone or more other selected kinases from the remaining group listed inTable 1 herein.

According to some embodiments, inhibitory profiles of MMI inhibitors invivo depend on dosages, routes of administration, and cell typesresponding to the inhibitors.

According to such embodiment, the pharmaceutical composition inhibitsless than 50% of the kinase activity of the other selected kinase(s).According to such embodiment, the pharmaceutical composition inhibitsless than 65% of the kinase activity of the other selected kinase(s).According to another embodiment, the pharmaceutical composition inhibitsless than 50% of the kinase activity of the other selected kinase(s).According to another embodiment, the pharmaceutical composition inhibitsless than 40% of the kinase activity of the other selected kinase(s).According to another embodiment, the pharmaceutical composition inhibitsless than 20% of the kinase activity of the other selected kinase(s).According to another embodiment, the pharmaceutical composition inhibitsless than 15% of the kinase activity of the other selected kinase(s).According to another embodiment, the pharmaceutical composition inhibitsless than 10% of the kinase activity of the other selected kinase(s).According to another embodiment, the pharmaceutical composition inhibitsless than 5% of the kinase activity of the other selected kinase(s).According to another embodiment, the pharmaceutical compositionincreases the kinase activity of the other selected kinases.

According to the embodiments of the immediately preceding paragraph, theone or more other selected kinase that is not substantially inhibited isselected from the group of Ca²⁺/calmodulin-dependent protein kinase II(CaMKII, including its subunit CaMKIIδ), Proto-oncogeneserine/threonine-protein kinase (PIM-1), cellular-Sarcoma (c-SRC),Spleen Tyrosine Kinase (SYK), C-src Tyrosine Kinase (CSK), andInsulin-like Growth Factor 1 Receptor (IGF-1R).

According to some embodiments, the pharmaceutical composition furthercomprises at least one additional therapeutic agent.

According to some such embodiments, examples of such additionaltherapeutic agents include, without limitation, purified bovine Type Vcollagens (e.g., IW-001; ImmuneWorks; United Therapeutics), IL-13receptor antagonists (e.g., QAX576; Novartis), protein tyrosine kinaseinhibitors (e.g., imatinib (Gleevec®); Craig Daniels/Novartis),endothelial receptor antagonists (e.g., ACT-064992 (macitentan);Actelion), dual endothelin receptor antagonists (e.g., bosentan(Tracleer®); Actelion), prostacyclin analogs (inhaled iloprost (e.g.,Ventavis®); Actelion), anti-CTGF monoclonal antibodies (e.g., FG-3019),endothelin receptor antagonists (A-selective) (e.g., ambrisentan(Letairis®), Gilead), AB0024 (Arresto), lysyl oxidase-like 2 (LOXL2)monoclonal antibodies (e.g., GS-6624 (formerly AB0024); Gilead), c-JunN-terminal kinase (JNK) inhibitors (e.g., CC-930; Celgene), Pirfenidone(e.g., Esbriet® (InterMune), Pirespa® (Shionogi)), IFN-γ1b (e.g.,Actimmune®; InterMune), pan-neutralizing IgG4 human antibodies againstall three TGF-β isoforms (e.g., GC1008; Genzyme), TGF-β activationinhibitors (e.g., Stromedix (STX-100)), recombinant human Pentraxin-2protein (rhPTX-2) (e.g., PRM151; Promedior), bispecific IL4/IL13antibodies (e.g., SAR156597; Sanofi), humanized monoclonal antibodiestargeting integrin αvβ6 (BIBF 1120; Boehringer Ingelheim),N-acetylcysteine (Zambon SpA), Sildenafil (Viagra®), TNF antagonists(e.g., etanercept (Enbrel®); Pfizer), glucocorticoids (e.g., prednisone,budesonide, mometasone furoate, fluticasone propionate, and fluticasonefuroate), bronchodilators (e.g., leukotriene modifers (e.g., Montelukast(SINGUAIR®)), anticholingertic bronchodilators (e.g., Ipratropiumbromide and Tiotropium), short-acting β2-agonists (e.g., isoetharinemesylate (Bronkometer®), adrenalin, salbutanol/albuterol, andterbutaline), long-acting β2-agonists (e.g., salmeterol, formoterol,indecaterol (Onbrez®), sofosbuvir (Sovaldi®), an HCV boosted proteaseinhibitor (ABT-450, AbbVie), a nonnucleoside NS5B inhibitor (dasabuvir,ABT-333, AbbVie), an NS5a inhibitor (ombitasvir, ABT-267, AbbVie),ABT-450/r (ABT-450 with ritonavir), ABT-450 co-formulated with ABT-267,ABT-450 formulated with sofosbuvir, ribavirin, and combinations thereof.

According to some other embodiments, the additional therapeutic agentcomprises a bronchodilator including, but not limited to, a leukotrienemodifier, an anticholinergic bronchodilator, a β2-agonist, or acombination thereof.

According to another embodiment, the additional therapeutic agentcomprises a corticosteroid including, but not limited to, prednisone,budesonide, mometasone, beclemethasone, or a combination thereof.

According to some other embodiments, the additional therapeutic agent isan anti-inflammatory agent.

According to some such embodiments, the anti-inflammatory agent is anonsteroidal anti-inflammatory agent. The term “non-steroidalanti-inflammatory agent” as used herein refers to a large group ofagents that are aspirin-like in their action, including, but not limitedto, ibuprofen (Advil®), naproxen sodium (Aleve®), and acetaminophen(Tylenol®). Additional examples of non-steroidal anti-inflammatoryagents that are usable in the context of the described inventioninclude, without limitation, oxicams, such as piroxicam, isoxicam,tenoxicam, sudoxicam, and CP-14,304; disalcid, benorylate, trilisate,safapryn, solprin, diflunisal, and fendosal; acetic acid derivatives,such as diclofenac, fenclofenac, indomethacin, sulindac, tolmetin,isoxepac, furofenac, tiopinac, zidometacin, acematacin, fentiazac,zomepirac, clindanac, oxepinac, felbinac, and ketorolac; fenamates, suchas mefenamic, meclofenamic, flufenamic, niflumic, and tolfenamic acids;propionic acid derivatives, such as benoxaprofen, flurbiprofen,ketoprofen, fenoprofen, fenbufen, indopropfen, pirprofen, carprofen,oxaprozin, pranoprofen, miroprofen, tioxaprofen, suprofen, alminoprofen,and tiaprofenic; pyrazoles, such as phenylbutazone, oxyphenbutazone,feprazone, azapropazone, and trimethazone. Mixtures of thesenon-steroidal anti-inflammatory agents also may be employed, as well asthe dermatologically acceptable salts and esters of these agents. Forexample, etofenamate, a flufenamic acid derivative, is particularlyuseful for topical application.

According to another embodiment, the nonsteroidal anti-inflammatoryagent comprises Transforming Growth Factor-β3 (TGF-β3), an anti-TumorNecrosis Factor-alpha (TNF-α) agent, or a combination thereof.

According to another embodiment, the anti-inflammatory agent is asteroidal anti-inflammatory agent. The term “steroidal anti-inflammatoryagent”, as used herein, refer to any one of numerous compoundscontaining a 17-carbon 4-ring system and includes the sterols, varioushormones (as anabolic steroids), and glycosides. Representative examplesof steroidal anti-inflammatory drugs include, without limitation,corticosteroids such as hydrocortisone, hydroxyltriamcinolone,alpha-methyl dexamethasone, dexamethasone-phosphate, beclomethasonedipropionates, clobetasol valerate, desonide, desoxymethasone,desoxycorticosterone acetate, dexamethasone, dichlorisone,diflucortolone valerate, fluadrenolone, fluclorolone acetonide,flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortinebutylesters, fluocortolone, fluprednidene (fluprednylidene) acetate,flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisonebutyrate, methylprednisolone, triamcinolone acetonide, cortisone,cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate,fluradrenolone, fludrocortisone, diflorosone diacetate, fluradrenoloneacetonide, medrysone, amcinafel, amcinafide, betamethasone and thebalance of its esters, chloroprednisone, chlorprednisone acetate,clocortelone, clescinolone, dichlorisone, diflurprednate, flucloronide,flunisolide, fluoromethalone, fluperolone, fluprednisolone,hydrocortisone valerate, hydrocortisone cyclopentylpropionate,hydrocortamate, meprednisone, paramethasone, prednisolone, prednisone,beclomethasone dipropionate, triamcinolone, and mixtures thereof.

According to another embodiment, the steroidal anti-inflammatory agentcomprises at least one corticosteroid selected from the group consistingof prednisone, budesonide, mometasone, beclemethasone, and a combinationthereof.

According to another embodiment, the additional therapeutic agentcomprises a xanthine or xanthine derivative, such as methylxanthine.

According to another embodiment, the additional therapeutic agentcomprises a neutrophil elastase inhibitor.

According to another embodiment, the additional therapeutic agent is atleast one neutrophil elastase inhibitor, including, but not limited to,ICI 200355, ONO-5046, MR-889, L-694,458, CE-1037, GW-311616, TEI-8362,ONO-6818, AE-3763, FK-706, ICI-200,880, ZD-0892, ZD-8321, and acombination thereof.

According to another embodiment, the additional therapeutic agentcomprises at least one phosphodiesterase inhibitor, including, but notlimited to, phosphodiesterase 4 inhibitor. Examples of phosphodiesterase4 inhibitors include, but are not limited to, roflumilast, cilomilast ora combination thereof.

According to another embodiment, the additional therapeutic agent is ananalgesic agent. According to some embodiments, the analgesic agentrelieves pain by elevating the pain threshold without disturbingconsciousness or altering other sensory modalities. According to somesuch embodiments, the analgesic agent is a non-opioid analgesic.“Non-opioid analgesics” are natural or synthetic substances that reducepain but are not opioid analgesics. Examples of non-opioid analgesicsinclude, but are not limited to, etodolac, indomethacin, sulindac,tolmetin, nabumetone, piroxicam, acetaminophen, fenoprofen,flurbiprofen, ibuprofen, ketoprofen, naproxen, naproxen sodium,oxaprozin, aspirin, choline magnesium trisalicylate, diflunisal,meclofenamic acid, mefenamic acid, and phenylbutazone. According to someother embodiments, the analgesic is an opioid analgesic. “Opioidanalgesics”, “opioid”, or “narcotic analgesics” are natural or syntheticsubstances that bind to opioid receptors in the central nervous system,producing an agonist action. Examples of opioid analgesics include, butare not limited to, codeine, fentanyl, hydromorphone, levorphanol,meperidine, methadone, morphine, oxycodone, oxymorphone, propoxyphene,buprenorphine, butorphanol, dezocine, nalbuphine, and pentazocine.

According to another embodiment, the additional therapeutic agent is ananti-infective agent. According to another embodiment, theanti-infective agent is an antibiotic agent. The term “antibiotic agent”as used herein means any of a group of chemical substances having thecapacity to inhibit the growth of, or to destroy bacteria and othermicroorganisms, used chiefly in the treatment of infectious diseases.Examples of antibiotic agents include, but are not limited to,Penicillin G; Methicillin; Nafcillin; Oxacillin; Cloxacillin;Dicloxacillin; Ampicillin; Amoxicillin; Ticarcillin; Carbenicillin;Mezlocillin; Azlocillin; Piperacillin; Imipenem; Aztreonam; Cephalothin;Cefaclor; Cefoxitin; Cefuroxime; Cefonicid; Cefmetazole; Cefotetan;Cefprozil; Loracarbef; Cefetamet; Cefoperazone; Cefotaxime; Ceftizoxime;Ceftriaxone; Ceftazidime; Cefepime; Cefixime; Cefpodoxime; Cefsulodin;Fleroxacin; Nalidixic acid; Norfloxacin; Ciprofloxacin; Ofloxacin;Enoxacin; Lomefloxacin; Cinoxacin; Doxycycline; Minocycline;Tetracycline; Amikacin; Gentamicin; Kanamycin; Netilmicin; Tobramycin;Streptomycin; Azithromycin; Clarithromycin; Erythromycin; Erythromycinestolate; Erythromycin ethyl succinate; Erythromycin glucoheptonate;Erythromycin lactobionate; Erythromycin stearate; Vancomycin;Teicoplanin; Chloramphenicol; Clindamycin; Trimethoprim;Sulfamethoxazole; Nitrofurantoin; Rifampin; Mupirocin; Metronidazole;Cephalexin; Roxithromycin; Co-amoxiclavuanate; combinations ofPiperacillin and Tazobactam; and their various salts, acids, bases, andother derivatives. Anti-bacterial antibiotic agents include, but are notlimited to, penicillins, cephalosporins, carbacephems, cephamycins,carbapenems, monobactams, aminoglycosides, glycopeptides, quinolones,tetracyclines, macrolides, and fluoroquinolones.

According to another embodiment, the pharmaceutical composition inhibitsinflammation occurring in a lung of the subject. According to anotherembodiment, the inflammation is an acute inflammation. According toanother embodiment, the inflammation is a chronic inflammation.According to another embodiment, the inflammation is mediated by TumorNecrosis Factor-alpha (TNF-α). According to another embodiment, theinflammation is mediated by Interleukin-6 (IL-6). According to anotherembodiment, the inflammation is mediated by Interleukin-1β (IL-1β).

According to another embodiment, the pharmaceutical compositionmodulates an amount of Tumor Necrosis Factor-alpha (TNF-α) in the lung,compared to a control. According to another embodiment, thepharmaceutical composition modulates the amount of Interleukin-6 (IL-6)in the lung, compared to a control. According to another embodiment, thepharmaceutical composition modulates the amount of Interleukin-1β(IL-1β) in the lung, compared to a control.

According to another embodiment, the pharmaceutical composition inhibitsan activity of Heat Shock 27 kDa protein 1 (HSPB1). According to anotherembodiment, the activity of HSPB1 inhibited by the pharmaceuticalcomposition is an aberrant induction of fibroblast proliferation.According to another embodiment, the activity of HSPB1 inhibited by thepharmaceutical composition is an aberrant induction of myofibroblastdifferentiation. According to another embodiment, the activity of HSPB1inhibited by the pharmaceutical composition is a deposition of anextracellular matrix protein into a pulmonary interstitium. According toanother embodiment, the extracelluar matrix protein is collagen.According to another embodiment, the activity of HSPB1 inhibited by thepharmaceutical composition is a promotion of fibrotic loci formation.According to another embodiment, the activity of HSPB1 inhibited by thepharmaceutical composition is an increase of a myofibroblast contractileactivity. According to another embodiment, the activity of HSPB1inhibited by the pharmaceutical composition is a promotion ofmyofibroblast attachment to extracellular matrix.

According to some embodiments, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) has a substantialsequence identity to amino acid sequence YARAAARQARAKALARQLGVAA (SEQ IDNO: 1).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) has at least 70percent sequence identity to amino acid sequence YARAAARQARAKALARQLGVAA(SEQ ID NO: 1). According to another embodiment, the functionalequivalent of the polypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) hasat least 80 percent sequence identity to amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1). According to another embodiment,the functional equivalent of the polypeptide YARAAARQARAKALARQLGVAA (SEQID NO: 1) has at least 90 percent sequence identity to amino acidsequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1). According to anotherembodiment, the functional equivalent of the polypeptideYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) has at least 95 percent sequenceidentity to amino acid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence FAKLAARLYRKALARQLGVAA (SEQ ID NO: 3).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence KAFAKLAARLYRKALARQLGVAA (SEQ ID NO: 4).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence YARAAARQARAKALARQLAVA (SEQ ID NO: 5).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence YARAAARQARAKALARQLGVA (SEQ ID NO: 6).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence HRRIKAWLKKIKALARQLGVAA (SEQ ID NO: 7).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence YARAAARQARAKALNRQLAVAA (SEQ ID NO: 26).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence YARAAARQARAKALNRQLAVA (SEQ ID NO: 27).

According to some other embodiments, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is a fusion proteincomprising a first polypeptide operatively linked to a secondpolypeptide, wherein the first polypeptide is of amino acid sequenceYARAAARQARA (SEQ ID NO: 11), and the second polypeptide comprises atherapeutic domain whose sequence has substantial identity to amino acidsequence KALARQLGVAA (SEQ ID NO: 2).

According to some such embodiments, the second polypeptide has at least70 percent sequence identity to amino acid sequence KALARQLGVAA (SEQ IDNO: 2).

According some other embodiments, the second polypeptide has at least 80percent sequence identity to amino acid sequence KALARQLGVAA (SEQ ID NO:2). According to some other embodiments, the second polypeptide has atleast 90 percent sequence identity to amino acid sequence KALARQLGVAA(SEQ ID NO: 2). According to some other embodiments, the secondpolypeptide has at least 95 percent sequence identity to amino acidsequence KALARQLGVAA (SEQ ID NO: 2).

According to some embodiments, the second polypeptide is a polypeptideof amino acid sequence KALARQLAVA (SEQ ID NO: 8).

According to another embodiment, the second polypeptide is a polypeptideof amino acid sequence KALARQLGVA (SEQ ID NO: 9).

According to another embodiment, the second polypeptide is a polypeptideof amino acid sequence KALARQLGVAA (SEQ ID NO: 10).

According to another embodiment, the second polypeptide is a polypeptideof amino acid sequence KALNRQLAVAA (SEQ ID NO: 28).

According to another embodiment, the second polypeptide is a polypeptideof amino acid sequence YKALNRQLAVA (SEQ ID NO: 29).

According to some other embodiments, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is a fusion proteincomprising a first polypeptide operatively linked to a secondpolypeptide, wherein the first polypeptide comprises a cell penetratingpeptide functionally equivalent to YARAAARQARA (SEQ ID NO: 11), and thesecond polypeptide is of amino acid sequence KALARQLGVAA (SEQ ID NO: 2).

According to another embodiment, the first polypeptide is a polypeptideof amino acid sequence WLRRIKAWLRRIKA (SEQ ID NO: 12). According toanother embodiment, the first polypeptide is a polypeptide of amino acidsequence WLRRIKA (SEQ ID NO: 13). According to another embodiment, thefirst polypeptide is a polypeptide of amino acid sequence YGRKKRRQRRR(SEQ ID NO: 14). According to another embodiment, the first polypeptideis a polypeptide of amino acid sequence WLRRIKAWLRRI (SEQ ID NO: 15).According to another embodiment, the first polypeptide is a polypeptideof amino acid sequence FAKLAARLYR (SEQ ID NO: 16). According to anotherembodiment, the first polypeptide is a polypeptide of amino acidsequence KAFAKLAARLYR (SEQ ID NO: 17). According to another embodiment,the first polypeptide is a polypeptide of amino acid sequenceHRRIKAWLKKI (SEQ ID NO: 18).

According to another aspect, the described invention also provides anisolated nucleic acid that encodes a protein sequence with at least 70%amino acid sequence identity to amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1). According to some embodiments,the isolated nucleic acid encodes a protein sequence with at least 80%amino acid sequence identity to amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1). According to some otherembodiments, the isolated nucleic acid encodes a protein sequence withat least 90% amino acid sequence identity to amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1). According to some otherembodiments, the isolated nucleic acid encodes a protein sequence withat least 95% amino acid sequence identity to amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1).

According to some other embodiments, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 100 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.00001 mg/kg body weight to about 100 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.0001 mg/kg body weight to about 100 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.001 mg/kg body weight to about 10 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.01 mg/kg body weight to about 10 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.1 mg/kg (or 100 μg/kg) body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 1 mg/kg body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 10 mg/kg body weight to about 100mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 2 mg/kg body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 3 mg/kg body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 4 mg/kg body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 5 mg/kg body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 60 mg/kg body weight to about 100mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 70 mg/kg body weight to about 100mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 80 mg/kg body weight to about 100mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 90 mg/kg body weight to about 100mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitor peptide of the pharmaceuticalcomposition is of an amount from about 0.000001 mg/kg body weight toabout 90 mg/kg body weight. According to another embodiment, thetherapeutic amount of the therapeutic inhibitor peptide of thepharmaceutical composition is of an amount from about 0.000001 mg/kgbody weight to about 80 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 70 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 60 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 50 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 40 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideis of an amount from about 0.000001 mg/kg body weight to about 30 mg/kgbody weight. According to another embodiment, the therapeutic amount ofthe therapeutic inhibitor peptide of the pharmaceutical composition isof an amount from about 0.000001 mg/kg body weight to about 20 mg/kgbody weight. According to another embodiment, the therapeutic amount ofthe therapeutic inhibitor peptide of the pharmaceutical composition isof an amount from about 0.000001 mg/kg body weight to about 10 mg/kgbody weight. According to another embodiment, the therapeutic amount ofthe therapeutic inhibitor peptide of the pharmaceutical composition isof an amount from about 0.000001 mg/kg body weight to about 1 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.1 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.1 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.01 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.001 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.0001 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.00001 mg/kg bodyweight.

According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 1 μg/kg/day to 25 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 1 μg/kg/day to 2 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 2 μg/kg/day to 3 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 3 μg/kg/day to 4 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical ranges from 4μg/kg/day to 5 μg/kg/day. According to some other embodiments, thetherapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 5 μg/kg/day to 6 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 6 μg/kg/day to 7 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 7 μg/kg/day to 8 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 8 μg/kg/day to 9 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 9 μg/kg/day to 10 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 1 μg/kg/day to 5 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 5 μg/kg/day to 10 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 10 μg/kg/day to 15 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 15 μg/kg/day to 20 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 25 μg/kg/day to 30 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 30 μg/kg/day to 35 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 35 μg/kg/day to 40 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 40 μg/kg/day to 45 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 45 μg/kg/day to 50 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 50 μg/kg/day to 55 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 55 μg/kg/day to 60 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 60 μg/kg/day to 65 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 65 μg/kg/day to 70 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 70 μg/kg/day to 75 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 80 μg/kg/day to 85 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 85 μg/kg/day to 90 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 90 μg/kg/day to 95 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 95 μg/kg/day to 100 μg/kg/day.

According to another embodiment, the therapeutic dose of the therapeuticinhibitor peptide of the pharmaceutical composition is 1 μg/kg/day.

According to another embodiment, the therapeutic dose of the therapeuticinhibitor peptide of the pharmaceutical composition is 2 μg/kg/day.

According to another embodiment, the therapeutic dose of the therapeuticinhibitor peptide of the pharmaceutical composition is 5 μg/kg/day.

According to another embodiment, the therapeutic dose of the therapeuticinhibitor peptide of the pharmaceutical composition is 10 μg/kg/day.

According to some embodiments, the polypeptide of the inventioncomprises D-amino acids (which are resistant to L-amino acid-specificproteases in vivo), a combination of D- and L-amino acids, and various“designer” amino acids (e.g., β-methyl amino acids, C-α-methyl aminoacids, and N-α-methyl amino acids, etc.) to convey special properties.Examples of synthetic amino acid substitutions include ornithine forlysine, and norleucine for leucine or isoleucine.

According to some embodiments, the polypeptide may be linked to othercompounds to promote an increased half-life in vivo, such aspolyethylene glycol or dextran. Such linkage can be covalent ornon-covalent as is understood by those of skill in the art. According tosome other embodiments, the polypeptide may be encapsulated in a micellesuch as a micelle made ofpoly(ethyleneglycol)-block-poly(polypropylenglycol) orpoly(ethyleneglycol)-block-polylactide. According to some otherembodiments, the polypeptide may be encapsulated in degradable nano- ormicro-particles composed of degradable polyesters including, but notlimited to, polylactic acid, polyglycolide, and polycaprolactone.

According to another embodiment, the polypeptide may be prepared in asolid form (including granules, powders or suppositories) or in a liquidform (e.g., solutions, suspensions, or emulsions).

According to another embodiment, the compositions of the describedinvention may be in the form of a dispersible dry powder for delivery byinhalation or insufflation (either through the mouth or through thenose, respectively). Dry powder compositions may be prepared byprocesses known in the art, such as lyophilization and jet milling, asdisclosed in International Patent Publication No. WO 91/16038 and asdisclosed in U.S. Pat. No. 6,921,527, the disclosures of which areincorporated by reference. The composition of the described invention isplaced within a suitable dosage receptacle in an amount sufficient toprovide a subject with a unit dosage treatment. The dosage receptacle isone that fits within a suitable inhalation device to allow for theaerosolization of the dry powder composition by dispersion into a gasstream to form an aerosol and then capturing the aerosol so produced ina chamber having a mouthpiece attached for subsequent inhalation by asubject in need of treatment. Such a dosage receptacle includes anycontainer enclosing the composition known in the art such as gelatin orplastic capsules with a removable portion that allows a stream of gas(e.g., air) to be directed into the container to disperse the dry powdercomposition. Such containers are exemplified by those shown in U.S. Pat.Nos. 4,227,522; 4,192,309; and 4,105,027. Suitable containers alsoinclude those used in conjunction with Glaxo's Ventolin® Rotohaler brandpowder inhaler or Fison's Spinhaler® brand powder inhaler. Anothersuitable unit-dose container which provides a superior moisture barrieris formed from an aluminum foil plastic laminate. Thepharmaceutical-based powders is filled by weight or by volume into thedepression in the formable foil and hermetically sealed with a coveringfoil-plastic laminate. Such a container for use with a powder inhalationdevice is described in U.S. Pat. No. 4,778,054 and is used with Glaxo'sDiskhaler® (U.S. Pat. Nos. 4,627,432; 4,811,731; and 5,035,237). All ofthese references are incorporated herein by reference in theirentireties.

According to another embodiment, the carrier of the composition of thedescribed invention includes a release agent, such as a sustainedrelease or delayed release carrier. In such embodiments, the carrier canbe any material capable of sustained or delayed release of thepolypeptide to provide a more efficient administration, e.g., resultingin less frequent and/or decreased dosage of the polypeptide, improvingease of handling, and extending or delaying effects on diseases,disorders, conditions, syndromes, and the like, being treated, preventedor promoted. Non-limiting examples of such carriers include liposomes,microsponges, microspheres, or microcapsules of natural and syntheticpolymers and the like. Liposomes may be formed from a variety ofphospholipids, including, but not limited to, cholesterol, stearylaminesor phosphatidylcholines.

Methods for synthesis and preparation of small peptides are well knownin the art and are disclosed, for example, in U.S. Pat. Nos. 5,352,461;5,503,852; 6,071,497; 6,331,318; 6,428,771 and U.S. Publication No.20060040953. U.S. Pat. Nos. 6,444,226 and 6,652,885 describe preparingand providing microparticles of diketopiperazines in aqueous suspensionto which a solution of active agent is added in order to bind the activeagent to the particle. These patents further describe a method ofremoving a liquid medium by lyophilization to yield microparticlescomprising an active agent. Altering the solvent conditions of suchsuspension to promote binding of the active agent to the particle isdisclosed in U.S. application Ser. Nos. 60/717,524; 11/532,063; and11/532,065; U.S. Pat. No. 6,440,463; and U.S. application Ser. Nos.11/210,709 and 11/208,087. Each of these patents and patent applicationsis incorporated by reference herein.

In some embodiments, MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) andits functional equivalents of the present invention can be dried by amethod of spraying drying as disclosed in, for example, U.S. applicationSer. No. 11/678,046 (incorporated by reference herein).

In yet another embodiment, the polypeptide of the invention may beapplied in a variety of solutions. A suitable formulation is sterile,dissolves sufficient amounts of the polypeptides, and is not harmful forthe proposed application. For example, the compositions of the describedinvention may be formulated as aqueous suspensions wherein the activeingredient(s) is (are) in admixture with excipients suitable for themanufacture of aqueous suspensions.

Such excipients include, without limitation, suspending agents (e.g.,sodium carboxymethylcellulose, methylcellulose,hydroxy-propylmethylcellulose, sodium alginate, polyvinylpyrrolidone,gum tragacanth, and gum acacia), dispersing or wetting agents including,a naturally-occurring phosphatide (e.g., lecithin), or condensationproducts of an alkylene oxide with fatty acids (e.g., polyoxyethylenestearate), or condensation products of ethylene oxide with long chainaliphatic alcohols (e.g., heptadecaethyl-eneoxycetanol), or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand a hexitol (e.g., polyoxyethylene sorbitol monooleate), orcondensation products of ethylene oxide with partial esters derived fromfatty acids and hexitol anhydrides (e.g., polyethylene sorbitanmonooleate).

Compositions of the described invention also may be formulated as oilysuspensions by suspending the active ingredient in a vegetable oil(e.g., arachis oil, olive oil, sesame oil or coconut oil) or in amineral oil (e.g., liquid paraffin). The oily suspensions may contain athickening agent (e.g., beeswax, hard paraffin or cetyl alcohol).

Compositions of the described invention also may be formulated in theform of dispersible powders and granules suitable for preparation of anaqueous suspension by the addition of water. The active ingredient insuch powders and granules is provided in admixture with a dispersing orwetting agent, suspending agent, and one or more preservatives. Suitabledispersing or wetting agents and suspending agents are exemplified bythose already mentioned above. Additional excipients also may bepresent.

According to some embodiments, the dry powder is produced by a spraydrying process.

According to some other embodiments, the dry powder is produced bymicronization

According to another embodiment, the dry powder comprises microparticleswith Mass Median Aerodynamic Diameter (MMAD) of 1 to 5 microns.

According to another embodiment, the dry powder comprises microparticleswith Mass Median Aerodynamic Diameter (MMAD) of about 2 micron.

According to another embodiment, the pharmaceutical composition ispackaged in an inhalation device, including, for example, but notlimited to a nebulizer, a metered-dose inhaler (MDI), and a dry powderinhaler (DPI).

According to some other embodiments, the pharmaceutical composition is aliquid for aerosolized delivery using a nebulizer. According to somesuch embodiments, the flow-rate of the pharmaceutical composition is atleast 0.3 ml/min, and the pharmaceutical composition is delivered as 2mm particles, with distribution into deepest alveoli.

Compositions of the described invention also may be in the form of anemulsion. An emulsion is a two-phase system prepared by combining twoimmiscible liquid carriers, one of which is disbursed uniformlythroughout the other and consists of globules that have diameters equalto or greater than those of the largest colloidal particles. The globulesize is critical and must be such that the system achieves maximumstability. Usually, separation of the two phases will not occur unless athird substance, an emulsifying agent, is incorporated. Thus, a basicemulsion contains at least three components, the two immiscible liquidcarriers and the emulsifying agent, as well as the active ingredient.Most emulsions incorporate an aqueous phase into a non-aqueous phase (orvice versa). However, it is possible to prepare emulsions that arebasically non-aqueous, for example, anionic and cationic surfactants ofthe non-aqueous immiscible system glycerin and olive oil. Thus, thecompositions of the invention may be in the form of an oil-in-wateremulsion. The oily phase may be a vegetable oil, for example, olive oilor arachis oil, or a mineral oil, for example a liquid paraffin, or amixture thereof. Suitable emulsifying agents may be naturally-occurringgums, for example, gum acacia or gum tragacanth, naturally-occurringphosphatides, for example soy bean, lecithin, and esters or partialesters derived from fatty acids and hexitol anhydrides, for examplesorbitan monooleate, and condensation products of the partial esterswith ethylene oxide, for example, polyoxyethylene sorbitan monooleate.

According to some embodiments, the polypeptide of the describedinvention is chemically synthesized. Such a synthetic polypeptide,prepared using the well known techniques of solid phase, liquid phase,or peptide condensation techniques, or any combination thereof, mayinclude natural and unnatural amino acids. Amino acids used for peptidesynthesis may be standard Boc (N-α-amino protectedN-α-t-butyloxycarbonyl) amino acid resin with the standard deprotecting,neutralization, coupling and wash protocols of the original solid phaseprocedure of Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154), or thebase-labile N-α-amino protected 9-fluorenylmethoxycarbonyl (Fmoc) aminoacids first described by Carpino and Han (1972, J. Org. Chem.37:3403-3409). Both Fmoc and Boc N-α-amino protected amino acids can beobtained from Sigma, Cambridge Research Biochemical, or other chemicalcompanies familiar to those skilled in the art. In addition, thepolypeptide may be synthesized with other N-α-protecting groups that arefamiliar to those skilled in this art. Solid phase peptide synthesis maybe accomplished by techniques familiar to those in the art and provided,for example, in Stewart and Young, 1984, Solid Phase Synthesis, SecondEdition, Pierce Chemical Co., Rockford, Ill.; Fields and Noble, 1990,Int. J. Pept. Protein Res. 35:161-214, or using automated synthesizers,each incorporated by reference herein in its entirety.

II. Methods for Preventing or Treating Diseases Characterized byAberrant Fibroblast Proliferation and Collagen Deposition

According to another aspect, the described invention provides a methodfor treating a disease, condition, or process characterized by aberrantfibroblast proliferation and extracellular matrix deposition in a tissueof a subject, the method comprising:

administering to the subject a pharmaceutical composition comprising atherapeutic amount of a polypeptide of the amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) or a functional equivalentthereof, and a pharmaceutically acceptable carrier thereof, wherein thetherapeutic amount is effective to reduce the fibroblast proliferationand extracellular matrix deposition in the tissue of the subject.

According to one embodiment of the method, the disease or the conditionis Acute Lung Injury (ALI) or acute respiratory distress syndrome(ARDS).

According to another embodiment, the disease or the condition isradiation-induced fibrosis.

According to another embodiment, the disease or the condition istransplant rejection.

According to another embodiment, the tissue is a lung tissue.

According to another embodiment, the disease or the condition is aninterstitial lung disease.

According to another embodiment, the disease or the condition ispulmonary fibrosis.

According to another embodiment, the pulmonary fibrosis is idiopathicpulmonary fibrosis.

According to another embodiment, the pulmonary fibrosis is caused byadministration of bleomycin.

According to another embodiment, the pulmonary fibrosis results from anallergic reaction, inhalation of environmental particulates, smoking, abacterial infection, a viral infection, mechanical damage to a lung ofthe subject, lung transplantation rejection, an autoimmune disorder, agenetic disorder, or a combination thereof.

According to another embodiment, the tissue is a liver tissue.

According to another embodiment, the disease or the condition is liverfibrosis.

Methods for detecting, diagnosing and assessing treatment of hepaticfibrosis include, but are not limited to, biopsy, imaging analysis andblood tests. Non-limiting examples of imaging analysis includeultrasonography, computed tomography (CT) and elastography. Examples ofelastography include, but are not limited to, ultrasound elastography,magnetic resonance elastogrphy, and the like. Blood tests include, butare not limited to, blood tests for the detection of biomarkers.Non-limiting examples of biomarkers include peptides, proteins, nucleicacids (e.g., DNA, RNA and mRNA), antibodies and genes.

Biomarkers for liver fibrosis include, but are not limited to, directbiomarkers (also know as class I fibrosis markers); cytokines andchemokines; and indirect biomarkers (also known as class II fibrosismarkers).

Ultrasonography is an imaging technique in which deep structures of thebody are visualized by recording the reflections (i.e., echoes) ofultrasonic waves directed into the tissues. Frequencies in the range of1 million to 10 million hertz are used in diagnostic ultrasonography.

The lower frequencies provide a greater depth of penetration and areused to examine abdominal organs. Frequencies in the upper range provideless penetration and are used predominantly to examine more superficialstructures, such as the eye. The basic principle of ultrasonography isthe same as that of depth-sounding in oceanographic studies of the oceanfloor. The ultrasonic waves are confined to a narrow beam that may betransmitted through, refracted, absorbed, or reflected by the mediumtoward which they are directed, depending on the nature of the surfacethey strike. In diagnostic ultrasonography, the ultrasonic waves areproduced by electrically stimulating a piezoelectric crystal called atransducer. As the beam strikes an interface or boundary between tissuesof varying acoustic impedance (e.g. muscle and blood) some of the soundwaves are reflected back to the transducer as echoes. The echoes arethen converted into electrical impulses that are displayed on anoscilloscope, presenting a ‘picture’ of the tissues under examination.Ultrasonography can be utilized in examination, for example, of theheart (echocardiography) and in identifying size and structural changesin organs in the abdominopelvic cavity (e.g., of the liver in liverfibrosis, liver cirrhosis, etc.). It is also useful in identifying anddistinguishing cancers and benign cysts.

Computerized tomography (CT) or computerized axial tomography (CAT) isan imaging technique in which an x-ray source and x-ray detector housedin a doughnut-shaped assembly move circularly around a patient who lieson a motorized table that is moved through the machine. Usually,multidetector scanners with 4 to 64 or more rows of detectors are usedin order to allow quicker scanning and higher-resolution images. Datafrom the detectors essentially represent a series of x-ray images takenfrom multiple angles around the patient. The images are not vieweddirectly but are sent to a computer, which quickly reconstructs theminto 2-dimensional images (tomograms) representing a slice of the bodyin any plane desired. Data can also be used to construct detailed3-dimensional images. For some CT scans, the table moves incrementallyand stops when each scan (slice) is taken. For other CT scans, the tablemoves continuously during scanning; because the patient is moving in astraight line and the detectors are moving in a circle, the series ofimages appear to be taken in a spiral fashion around the patient.Compared with plain x-rays, the tomographic slices of CT provide morespatial detail and can better differentiate between various soft-tissuedensities. Because it provides so much more information, CT is preferredto plain x-rays for imaging most intracranial, head and neck, spinal,intrathoracic, and intra-abdominal structures. Three-dimensional imagesof lesions can help surgeons plan surgery. CT is the most accurate studyfor detecting and localizing urinary calculi. CT may be done with orwithout IV contrast. Noncontrast CT is used to detect, for example,acute hemorrhage in the brain, urinary calculi, and lung nodules, aswell as to characterize bone fractures and other skeletal abnormalities.IV contrast is used, for example, to improve imaging of tumors,infection, inflammation, and trauma in soft tissues and to assess thevascular system, as when pulmonary embolism, aortic aneurysm, or aorticdissection is suspected.

Hepatic elastography is a method for estimating liver stiffness.Elastography can be accomplished by ultrasound or magnetic resonance.

Ultrasound Elastography (Also Called Transient Elastography)

A FibroScan device (EchoSens, Paris, France) uses a mild-amplitude,low-frequency (50-Hz) vibration transmitted through the liver. Itinduces an elastic shear wave that is detected by pulse-echoultrasonography as the wave propagates through the organ. The velocityof the wave correlates with tissue stiffness, i.e., the wave travelsfaster through denser, fibrotic tissue. Ultrasound elastography cansample a much larger area than liver biopsy, providing a betterunderstanding of the entire hepatic parenchyma. Moreover, it can berepeated often without risk.

Magnetic Resonance Elastography

Magnetic resonance elastography is similar to ultrasound elastography inthat it uses a vibration device to induce a shear wave in the liver.However, in this case, the wave is detected by a modified magneticresonance imaging machine, and a color-coded image is generated thatdepicts the wave velocity, and hence stiffness, throughout the organ. Inmagnetic resonance elastography, a value greater than 4.46 kPa indicatescirrhosis (and a value less than 4.13 indicates no cirrhosis) with ahigh degree of likelihood, and a value less than 2.84 excludes thelikelihood of significant fibrosis.

Direct (class I) biomarkers of liver fibrosis include, but are notlimited to, direct markers linked to matrix deposition and directmarkers linked to matrix degradation. Non-limiting examples of directmarkers linked to matrix deposition include, procollagen type 1 (PC1CP),procollagen type 3 (PCIIINP), type IV collagen, hyaluronic acid (HA),laminin and YXL-40 chondrex. Examples of direct markers linked to matrixdegradation include, but are not limited to, matrix metalloproteinase-1(MMP-1), matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9(MMP-9) and tissue inhibitors of matrix metalloproteinases (TIMPs)

Cytokine and chemokine biomarkers of liver fibrosis include, but are notlimited to, transforming growth factor-β (TGFβ), transforming growthfactor-α (TFGα) and platelet-derived growth factor (PDGF).

Indirect (class II) biomarkers of liver fibrosis include, but are notlimited to, aspartate aminotransferase/alanine aminotransferase(AST/ALT) ratio, PGA (prothrombin index, γ glutamyl transferase (GGT),and apolipoprotein A1), APRI (AST/platent count ratio), FibroSpect II(HA, TIMP-1, α2-macroglobulin), FibroTest/FibroSure (γ2 macroglobulin,γ2 globulin, γ globulin, apolipoprotein A1, GGT, total bilirubin),Fibrolndex (platelet count, AST, GGT), FibroMeter (platelet count, γ2macroglobulin, AST, age, prothrombin index, HA, blood urea nitrogen),Forns (age, platelet count, GGT, cholesterol levels), Hepascore (age,gender, bilirubin, GGT, HA, γ2 macroglobulin), FIB-4 (platelet count,ALT, AST, age), SHASTA index (HA, AST, albumin), Simple test (age,hyperglycemia, platelet count, albumin, AST/ALT, body mass index (BMI))and OELF/ELF (age, HA, N-terminal propeptide of type III collagen,TIMP-1).

According to another embodiment, the tissue is a kidney tissue.

According to another embodiment, the disease or condition is renalfibrosis.

Methods for detecting, diagnosing and assessing treatment of renalfibrosis include, but are not limited to, biopsy, imaging analysis andblood tests. Non-limiting examples of imaging analysis includehistological quantitive image analysis of pricrosirius red staining.Blood tests include, but are not limited to, blood tests for thedetection of biomarkers. Non-limiting examples of biomarkers includepeptides, proteins, nucleic acids (e.g., DNA, RNA and mRNA), antibodiesand genes. Renal fibrosis biomarkers include, but are not limited to,hydroxyproline, MCP-1, Collagen 1a1, Collagen 3a1, TGF-β, Fibronectin-1,α-SMA, connective tissue growth factor-1 (CTGF-1) and the like.

According to another embodiment, the tissue is a vascular tissue.

According to another embodiment, the disease or condition is vascularfibrosis.

Methods for detecting, diagnosing and assessing treatment of vascularfibrosis include, but are not limited to, biopsy, imaging analysis andblood tests. Non-limiting examples of imaging analysis includehistological quantitive image analysis of pricrosirius red staining,3′,3′-diaminobenzidine (DAB) and hematoxylin. Blood tests include, butare not limited to, blood tests for the detection of biomarkers.Non-limiting examples of biomarkers include peptides, proteins, nucleicacids (e.g., DNA, RNA and mRNA), antibodies and genes. Vascular fibrosisbiomarkers include, but are not limited to, hydroxyproline, MCP-1,Collagen 1a1, Collagen 3a1, TGF-β, Fibronectin-1, α-SMA, connectivetissue growth factor-1 (CTGF-1), PARAγ and the like.

According to another embodiment, the described invention utilizesisolated nucleic acids. Nucleic acids include, for example, DNA, RNA andmRNA. Nucleic acids can be isolated, for example, from tissues, cells,blood, serum, plasma, urine, saliva, semen and the like. Protocols andreagents for isolating nucleic acids are known. Non-limiting examples ofreagents used for nucleic acid isolation include guanidine thiocyanate,guanidine hydrochloride and guanidinium thiocyanate-phenol-chloroform;the proprietary formulation of this reagent is known as Trizol®.

According to another embodiment, the described invention utilizesmethods employing amplification of nucleic acids. Amplification ofnucleic acids is accomplished by Polymerase Chain Reaction (PCR).Non-limiting examples of PCR include conventional PCR, real-time PCR,quantitative PCR, quantitative real-time PCR, multiplex PCR,conventional reverse-transcriptase PCR (RT-PCR), real-time RT-PCR,quantitative RT-PCR, quantitative real-time RT-PCR, multiplex RT-PCR andthe like.

Primers for PCR amplification of target sequences (e.g., mRNA sequencesof TGFβ and α-SMA genes) can be designed based on the sequence of thetarget sequence, in accordance with standard procedures. Primersfunction to anneal and amplify a unique target sequence and asgenerators of a signal for detection and monitoring of an amplificationreaction. According to some embodiments, the primers are unlabeled (suchas in conventional PCR), while in other embodiments, the primers arelabeled, such as with a fluorescent moiety. Labeled primers can be ofany type, including those that are typically used in quantitative RT-PCRreactions, such as Scorpions, Molecular Beacons, and the like.

Probes may be provided in addition to primers. Probes that can be usedfor detection of amplification of the unique genomic sequences (e.g.,TaqMan® probes) can be designed to hybridize to a sequence between thetwo amplification primers, preferably within 5-15 bases of one of theprimer binding sites. Typically, probes are present in reaction mixturesin conjunction with primers or sets of primers for a particularamplification reaction, for example, an amplification of a unique targetsequence. However, probes may be provided as separate components, whichare separate from the primer(s) or other components of a reactionmixture.

The primers and probes are designed to have a typical size for primersand probes for use in PCR reactions. In general, the primers arerelatively short (about 10-30 bases in length) oligonucleotides, whilethe probes (e.g., TaqMan® probes) may be from about 15-35 bases inlength. The primers and probes are designed through a process thatincludes identification of unique sequences on a target nucleic acid,designing short oligonucleotides to amplify or detect those sequences,and synthesizing the oligonucleotides. Several characteristics may betaken into consideration when designing the primers and probe: e.g., theprobe melting temperature should be higher than the primer meltingtemperatures, and the distance between the 3′-end of one primer and the5′-end of the probe may be greater than 8 nucleotides. One of skill inthe art may select among such considerations and characteristics toprovide suitable primers and probes. Protocols for synthesis ofoligonucleotides are known to those skilled in the art. Any suitableprotocol may be used in synthesizing the primers and probes of theinvention.

Quantitative real-time RT-PCR is an accurate, precise, high throughputassay. Real-time PCR automates the process of quantitating reactionproducts for each sample in every cycle. According to some embodiments,real-time PCR systems rely upon the detection and quantitation of afluorescent reporter, the signal of which increases in direct proportionto the amount of PCR product in a reaction.

According to another embodiment, for example, the reporter is thedouble-stranded DNA-specific dye SYBR® Green (Molecular Probes), whichbinds double-stranded DNA, and upon excitation emits light. Thus, as aPCR product accumulates, fluorescence increases. The SYBR® Green(Molecular Probes, Eugene, Oreg.) system is one way to detect andquantitate PCR products in real time. The SYBR® Green dye binds, in asequence non-specific manner, to double-stranded nucleic acids. It thuscan be used for detection and quantitation of double-stranded productsproduced from single-stranded templates (e.g., mRNA). Other detectableprobes and primers, such as Amplifluor® probes, and DNAzymes, may beoptimized to be used for quantitative detection of amplificationproducts.

Alternatives to SYBR® Green include, but are not limited to, TaqMan®(Applied Biosystems, Foster City, Calif.) and molecular beacons, both ofwhich are hybridization probes relying on fluorescence resonance energytransfer (FRET) for quantitation. TaqMan® Probes are oligonucleotidesthat contain a fluorescent dye, typically on the 5′ base, and aquenching dye, typically located on the 3′ base. More specifically, forTaqMan® probes, when the probe is intact, the quencher quenches thesignal produced by the fluorescent label. However, upon binding of theprobe to the target sequence and subsequent digestion of the probe bythe 5′-3′ exonuclease activity of a polymerase, such as Taq polymerase,the fluorescent moiety is released from the quencher moiety, and adetectable signal, which is proportional to the amount of target nucleicacid being produced, is produced and can be monitored. According to oneembodiment, Taq polymerase is used in qRT-PCR due to its 5′-3′exonuclease activity, and it changes the fluorescence of the probes andallows amplification of CDR1 mRNA. TaqMan® probes rely on degradation bya polymerase to generate a detectable signal, while Scorpions® andMolecular Beacons rely on opening of a hairpin structure to provide adetectable signal Like TaqMan® probes, Scorpion® probes contain both afluorescent moiety and quenching moiety on a single probe. However,unlike TaqMan® probes, Scorpions® are not degraded during theamplification reaction. Rather, they are designed as primers foramplification reactions. Scorpion® primers are designed to form hairpinstructures in solution, which causes the fluorescent moiety and thequenching moiety to be in close proximity. Binding of the primers totarget nucleic acids unfolds the hairpin structure and moves thequenching moiety a sufficient distance away from the fluorescent moietythat detectable fluorescence is emitted.

Molecular beacons also contain fluorescent and quenching dyes, but FRETonly occurs when the quenching dye is directly adjacent to thefluorescent dye. Molecular beacons are designed to adopt a hairpinstructure while free in solution, bringing the fluorescent dye andquencher in close proximity. When a molecular beacon hybridizes to atarget, the fluorescent dye and quencher are separated, FRET does notoccur, and the fluorescent dye emits light upon irradiation.

Multiplexing of PCR reactions is common. Multiplexing allows aninvestigator to assay two or more different gene targets in a singlereaction through the use of multiple probes or primers, each specificfor its own target and each comprising a fluorescent moiety that emitsat a unique wavelength (as compared to the other probes). Multiplexingis possible with TaqMan® probes, Molecular Beacons, and Scorpions. Dueto its non-specific binding nature, SYBR® Green may not be amenable tomultiplexing.

Generally, a quantitative RT-PCR reaction is performed by one of twomethods: comparison to a standard curve or comparison of Ct values. Inthe first of these methods, a standard curve of amplification productsof a particular mRNA is made based on amplification of a series ofdifferent, known amounts of a pre-selected nucleic acid. Amplificationresults of reactions performed on a target nucleic acid are thencompared to the standard curve to obtain a quantity, and that quantitycan be extrapolated to an amount of the target in the original sample.While it is preferred to use an mRNA as the source for the standardcurve, the stability of mRNA is known to affect the validity of suchstandard curves, and overcoming or minimizing this problem has proved tobe difficult. To avoid the problems associated with using mRNA as asource for the standard curve, researchers have used DNA for generationof standard curves. While use of DNA overcomes the problems associatedwith use of mRNA, the mere fact that it avoids the problems creates yetanother problem, i.e., because DNA templates are relatively stable, andbecause amplification of DNA does not require a first-strand synthesisstep (which can be inefficient and variable across samples orpreparations), the standard curves produced from DNA sources often donot correlate accurately to the amount of mRNA in a test sample.

In the Ct comparison method for quantitating PCR products, expression ofa housekeeping gene (such as β-actin) is used as a standard againstwhich amplification of a target nucleic acid (e.g., TGFβ and α-SMA) iscompared. Often, in this method, a comparison of expression of thetarget nucleic acid under two different conditions is performed todetermine changes in expression patterns. This method avoids theproblems associated with instability of RNA or use of DNA as a controlthat is seen when using the classical standard curve method.

Controls are amplified in the same PCR reaction mixture as the targetsequence in an effort to quantitate PCR products and determine amountsof target nucleic acids in a sample. These controls are oftentranscripts of housekeeping genes. Such housekeeping genes include, butare not limited to, β-actin and GAPDH. The control is added to thereaction mix and co-amplified with the target nucleic acid. Fluorescentprobes specific for both are included in the mixture, and twoamplification curves are obtained. The relative expression of the targetnucleic acid with respect to the control is then determined. Using thistechnique, multiple, different samples can be compared for expression ofa target gene (e.g., TGFβ and α-SMA), with reference back to the samecontrol. Although adding a control to amplification reactions can be auseful alternative to other methods of quantitating expression levels,and can be a useful method for normalizing PCR reactions across samples,it does not allow one to determine absolute amounts of materials presentin the amplification reaction mixture or in the original sample. Rather,the results are qualitative or semi-quantitative, giving an idea only ofthe amount of one nucleic acid (e.g., the target) in comparison toanother (e.g., the control).

According to another embodiment, the described invention providesmethods that detect a protein in a sample obtained from a subject. Suchmethods of protein detection include, but are not limited to, Westernblot, immunohistochemistry, enzyme-linked immunosorbant assay (ELISA),radioimmunoassay and the like.

Methods that detect proteins may employ antibodies. Antibodies are serumproteins the molecules of which possess small areas of their surfacethat are complementary to small chemical groupings on their targets.These complementary regions (referred to as the antibody combining sitesor antigen binding sites) of which there are at least two per antibodymolecule, and in some types of antibody molecules ten, eight, or in somespecies as many as 12, may react with their corresponding complementaryregion on the antigen (the antigenic determinant or epitope) to linkseveral molecules of multivalent antigen together to form a lattice.

The basic structural unit of a whole antibody molecule consists of fourpolypeptide chains, two identical light (L) chains (each containingabout 220 amino acids) and two identical heavy (H) chains (each usuallycontaining about 440 amino acids). The two heavy chains and two lightchains are held together by a combination of noncovalent and covalent(disulfide) bonds. The molecule is composed of two identical halves,each with an identical antigen-binding site composed of the N-terminalregion of a light chain and the N-terminal region of a heavy chain. Bothlight and heavy chains usually cooperate to form the antigen bindingsurface.

Human antibodies show two kinds of light chains, κ and λ; individualmolecules of immunoglobulin generally are only one or the other. Innormal serum, 60% of the molecules have been found to have κdeterminants and 30 percent λ. Many other species have been found toshow two kinds of light chains, but their proportions vary. For example,in the mouse and rat, λ chains comprise but a few percent of the total;in the dog and cat, κ chains are very low; the horse does not appear tohave any κ chain; rabbits may have 5 to 40% λ, depending on strain andb-locus allotype; and chicken light chains are more homologous to λ thanκ.

In mammals, there are five classes of antibodies, IgA, IgD, IgE, IgG,and IgM, each with its own class of heavy chain—α (for IgA), δ (forIgD), ε (for IgE), γ (for IgG) and μ (for IgM). In addition, there arefour subclasses of IgG immunoglobulins (IgG1, IgG2, IgG3, IgG4) havingγ1, γ2, γ3, and γ4 heavy chains respectively. In its secreted form, IgMis a pentamer composed of five four-chain units, giving it a total of 10antigen binding sites. Each pentamer contains one copy of a J chain,which is covalently inserted between two adjacent tail regions.

All five immunoglobulin classes differ from other serum proteins in thatthey show a broad range of electrophoretic mobility and are nothomogeneous. This heterogeneity—that individual IgG molecules, forexample, differ from one another in net charge—is an intrinsic propertyof the immunoglobulins.

An antigenic determinant or epitope is an antigenic site on a molecule.Sequential antigenic determinants/epitopes essentially are linearchains. In ordered structures, such as helical polymers or proteins, theantigenic determinants/epitopes essentially would be limited regions orpatches in or on the surface of the structure involving amino acid sidechains from different portions of the molecule which could come close toone another. These are conformational determinants.

The principle of complementarity, which often is compared to the fittingof a key in a lock, involves relatively weak binding forces (hydrophobicand hydrogen bonds, van der Waals forces, and ionic interactions), whichare able to act effectively only when the two reacting molecules canapproach very closely to each other and indeed so closely that theprojecting constituent atoms or groups of atoms of one molecule can fitinto complementary depressions or recesses in the other.Antigen-antibody interactions show a high degree of specificity, whichis manifest at many levels. Brought down to the molecular level,specificity means that the combining sites of antibodies to an antigenhave a complementarity not at all similar to the antigenic determinantsof an unrelated antigen. Whenever antigenic determinants of twodifferent antigens have some structural similarity, some degree offitting of one determinant into the combining site of some antibodies tothe other may occur, and that this phenomenon gives rise tocross-reactions. Cross reactions are of major importance inunderstanding the complementarity or specificity of antigen-antibodyreactions. Immunological specificity or complementarity makes possiblethe detection of small amounts of impurities/contaminations amongantigens.

Monoclonal antibodies (mAbs) can be generated by fusing mouse spleencells from an immunized donor with a mouse myeloma cell line to yieldestablished mouse hybridoma clones that grow in selective media. Ahybridoma cell is an immortalized hybrid cell resulting from the invitro fusion of an antibody-secreting B cell with a myeloma cell. Invitro immunization, which refers to primary activation ofantigen-specific B cells in culture, is another well-established meansof producing mouse monoclonal antibodies. The term “humanized monoclonalantibodies” refers to monoclonal antibodies in which the complementaritydetermining regions, (“CDRs”), which fashion the antibody binding siteof a mouse monoclonal antibody, are replaced with a CDR of a humanprotein, while maintaining the framework and constant regions of themouse antibody.

Diverse libraries of immunoglobulin heavy (VH) and light (Vκ and Vλ)chain variable genes from peripheral blood lymphocytes also can beamplified by polymerase chain reaction (PCR) amplification. Genesencoding single polypeptide chains in which the heavy and light chainvariable domains are linked by a polypeptide spacer (single chain Fv orscFv) can be made by randomly combining heavy and light chain V-genesusing PCR. A combinatorial library then can be cloned for display on thesurface of filamentous bacteriophage by fusion to a minor coat proteinat the tip of the phage.

The technique of guided selection is based on human immunoglobulin Vgene shuffling with rodent immunoglobulin V genes. The method entails(i) shuffling a repertoire of human λ light chains with the heavy chainvariable region (VH) domain of a mouse monoclonal antibody reactive withan antigen of interest; (ii) selecting half-human Fabs on that antigen(iii) using the selected λ light chain genes as “docking domains” for alibrary of human heavy chains in a second shuffle to isolate clone Fabfragments having human light chain genes; (v) transfecting mouse myelomacells by electroporation with mammalian cell expression vectorscontaining the genes; and (vi) expressing the V genes of the Fabreactive with the antigen as a complete IgG1, λ antibody molecule in themouse myeloma

According to another embodiment, the described invention utilizesmultiplex bead technology (e.g., Luminex®) to detect protein and nucleicacid biomarkers. For example, color-coded polystyrene orsuperparamagnetic beads coated with analyte-specific antibodies (i.e.,for protein biomarker detection) or anti-TAG sequences (i.e., fornucleic acid biomarker detection) recognizing different targets aremixed together and incubated with biotin-labeled targets (i.e., proteinor nucleic acid biomarkers). Captured targets are subsequently detectedusing a streptavidin-phycoerythrin conjugate.

According to another embodiment, the disease or the condition is furthercharacterized by an inflammation in the tissue.

According to another embodiment, the inflammation is an acute or achronic inflammation.

According to another embodiment, the inflammation is mediated by atleast one cytokine selected from the group consisting of Tumor NecrosisFactor-alpha (TNF-α), Interleukin-6 (IL-6), and Interleukin-1β (IL-1β).

According to another embodiment, the pulmonary fibrosis is characterizedby at least one pathology selected from the group consisting of anaberrant deposition of an extracellular matrix protein in a pulmonaryinterstitium, an aberrant promotion of fibroblast proliferation in thelung, an aberrant induction of myofibroblast differentiation in thelung, and an aberrant promotion of attachment of myofibroblasts to anextracellular matrix compared to a normal healthy control subject.

According to another embodiment, the aberrant fibroblast proliferationand extracellular matrix deposition in the tissue is characterized by anaberrant activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2) in the tissue compared to the activity ofMitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2) in thetissue of a normal healthy control subject.

According to another embodiment, the aberrant fibroblast proliferationand extracellular matrix deposition in the tissue is evidenced by anaberrant amount or distribution of activated (phosphorylated)Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2) in thetissue compared to the amount or distribution of activatedMitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2) in thetissue of a normal healthy control subject.

According to another embodiment, the pulmonary fibrosis is characterizedby at least one pathology selected from the group consisting of anaberrant deposition of an extracellular matrix protein in a pulmonaryinterstitium, an aberrant promotion of fibroblast proliferation in thelung, an aberrant induction of differentiation of a population offibroblasts into a population of myofibroblasts in the lung, and anaberrant promotion of attachment of myofibroblasts to an extracellularmatrix compared to a normal healthy control subject.

According to another embodiment, the disease or condition is a chronicobstructive pulmonary disease (COPD). According to another embodiment,the chronic obstructive pulmonary disease (COPD) is caused by smoking.According to another embodiment, the chronic obstructive pulmonarydisease (COPD) is caused by environmental particulates. According toanother embodiment, the chronic obstructive pulmonary disease (COPD) iscaused by alpha-1 antitrypsin deficiency. According to anotherembodiment, the chronic obstructive pulmonary disease (COPD) is causedby a childhood respiratory infection.

According to another embodiment, the pulmonary fibrosis is characterizedby an abnormal activity of Heat Shock 27 kDa protein 1 (HSPB1) in a lungof the subject compared to a normal healthy control subject. Accordingto another embodiment, the abnormal activity of HSPB1 is an aberrantdeposition of an extracellular matrix protein in a pulmonaryinterstitium of the subject compared to a normal healthy controlsubject. According to another embodiment, the extracellular matrixprotein is collagen. According to another embodiment, the abnormalactivity of HSPB1 is an aberrant promotion of fibroblast proliferationin the lung compared to a normal healthy control subject. According toanother embodiment, the abnormal activity of HSPB1 is aberrant inductionof myofibroblast differentiation in the lung compared to a normalhealthy control subject. According to another embodiment, the abnormalactivity of HSPB1 is a promotion of fibrotic loci formation in the lungcompared to a normal healthy control subject. According to anotherembodiment, the abnormal activity of HSPB1 is an increase ofmyofibroblast contractile activity in the lung compared to a normalhealthy control subject. According to another embodiment, the abnormalactivity of HSPB1 is an aberrant promotion of myofibroblast attachmentto an extracellular matrix in the lung compared to a normal healthycontrol subject.

According to another embodiment, the pharmaceutical composition inhibitsinflammation occurring in a lung of the subject. According to anotherembodiment, the inflammation is an acute inflammation. According toanother embodiment, the inflammation is a chronic inflammation.According to another embodiment, the inflammation is mediated by TumorNecrosis Factor-alpha (TNF-α). According to another embodiment, theinflammation is mediated by interleukin-1β (IL-1β). According to anotherembodiment, the inflammation is mediated by interleukin-6 (IL-6).

According to another embodiment, the pharmaceutical compositionmodulates an amount of Tumor Necrosis Factor-alpha (TNF-α) in the lungof the subject, compared to an untreated control. According to anotherembodiment, the pharmaceutical composition modulates an amount ofinterleukin-1β (IL-1β) in the lung of the subject, compared to acontrol. According to another embodiment, the pharmaceutical compositionmodulates an amount of interleukin-6 (IL-6) in the lung of the subject,compared to a control.

According to another embodiment, the pharmaceutical composition inhibitsan abnormal activity of HSPB1 compared to a normal healthy controlsubject in a lung of the subject. According to another embodiment, theabnormal activity of HSPB1 is an aberrant deposition of an extracellularmatrix protein in a pulmonary interstitium compared to a normal healthycontrol subject. According to another embodiment, the extracellularmatrix protein is collagen. According to another embodiment, theabnormal activity of HSPB1 is an aberrant promotion of fibroblastproliferation in the lung compared to a normal healthy control subject.According to another embodiment, the abnormal activity of HSPB1 is anaberrant induction of fibroblast differentiation into myofibroblasts inthe lung compared to a normal healthy control subject. According toanother embodiment, the abnormal activity of HSPB1 is an aberrantpromotion of fibrotic loci formation compared to a normal healthycontrol subject. According to another embodiment, the abnormal activityof HSPB1 is an aberrant increase in contractile activity ofmyofibroblasts compared to a normal healthy control subject. Accordingto another embodiment, the myofibroblast contractile activity ischaracterized by an elevated level of alpha smooth muscle actin (α-SMA)compared to a normal healthy control subject. According to anotherembodiment, the myofibroblasts contractile activity is characterized byincreases in stress-fiber formation compared to a normal healthy controlsubject. According to another embodiment, the abnormal activity of HSPB1is aberrant promotion of myofibroblasts attachment to an extracellularmatrix compared to a normal healthy control subject.

According to one embodiment, the pharmaceutical composition inhibits akinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2 kinase). According to another embodiment, thepharmaceutical composition inhibits at least 50% of the kinase activityof MK2 kinase. According to another embodiment, the pharmaceuticalcomposition inhibits at least 65% of the kinase activity of MK2 kinase.According to another embodiment, the pharmaceutical composition inhibitsat least 75% of the kinase activity of MK2 kinase. According to anotherembodiment, the pharmaceutical composition inhibits at least 80% of thekinase activity of MK2 kinase. According to another embodiment, thepharmaceutical composition inhibits at least 85% of the kinase activityof MK2 kinase. According to another embodiment, the pharmaceuticalcomposition inhibits at least 90% of the kinase activity of MK2 kinase.According to another embodiment, the pharmaceutical composition inhibitsat least 95% of the kinase activity of MK2 kinase.

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 3 (MK3 kinase). According to another embodiment, thepharmaceutical composition further inhibits at least 50% of the kinaseactivity of MK3 kinase. According to another embodiment, thepharmaceutical composition further inhibits at least 65% of the kinaseactivity of MK3 kinase. According to another embodiment, thepharmaceutical composition further inhibits at least 70% of the kinaseactivity of MK3 kinase. According to another embodiment, thepharmaceutical composition further inhibits at least 75% of the kinaseactivity of MK3 kinase. According to another embodiment, thepharmaceutical composition further inhibits at least 80% of the kinaseactivity of MK3 kinase. According to another embodiment, thepharmaceutical composition further inhibits at least 85% of the kinaseactivity of MK3 kinase. According to another embodiment, thepharmaceutical composition further inhibits at least 90% of the kinaseactivity of MK3 kinase. According to another embodiment, thepharmaceutical composition further inhibits at least 95% of the kinaseactivity of MK3 kinase.

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of calcium/calmodulin-dependent protein kinase I(CaMKI). According to another embodiment, the pharmaceutical compositionfurther inhibits at least 50% of the kinase activity ofCa²⁺/calmodulin-dependent protein kinase I (CaMKI). According to anotherembodiment, the pharmaceutical composition further inhibits at least 65%of the kinase activity of Ca²⁺/calmodulin-dependent protein kinase I(CaMKI). According to another embodiment, the pharmaceutical compositionfurther inhibits at least 70% of the kinase activity ofCa²⁺/calmodulin-dependent protein kinase I (CaMKI). According to anotherembodiment, the pharmaceutical composition further inhibits at least 75%of the kinase activity of Ca²⁺/calmodulin-dependent protein kinase I(CaMKI). According to another embodiment, the pharmaceutical compositionfurther inhibits at least 80% of the kinase activity ofCa²⁺/calmodulin-dependent protein kinase I (CaMKI). According to anotherembodiment, the pharmaceutical composition further inhibits at least 85%of the kinase activity of Ca²⁺/calmodulin-dependent protein kinase I(CaMKI). According to another embodiment, the pharmaceutical compositionfurther inhibits at least 90% of the kinase activity ofCa²⁺/calmodulin-dependent protein kinase I (CaMKI). According to anotherembodiment, the pharmaceutical composition further inhibits at least 95%of the kinase activity of Ca²⁺/calmodulin-dependent protein kinase I(CaMKI).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of BDNF/NT-3 growth factors receptor (TrkB). Accordingto another embodiment, the pharmaceutical further inhibits at least 50%of the kinase activity of BDNF/NT-3 growth factors receptor (TrkB).According to another embodiment, the pharmaceutical further inhibits atleast 65% of the kinase activity of BDNF/NT-3 growth factors receptor(TrkB). According to another embodiment, the pharmaceutical furtherinhibits at least 70% of the kinase activity of BDNF/NT-3 growth factorsreceptor (TrkB). According to another embodiment, the pharmaceuticalfurther inhibits at least 75% of the kinase activity of BDNF/NT-3 growthfactors receptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2) and a kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 3 (MK3).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2) and a kinase activity of calcium/calmodulin-dependentprotein kinase I (CaMKI).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2) and a kinase activity of BDNF/NT-3 growth factorsreceptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2), a kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 3 (MK3), a kinase activity ofcalcium/calmodulin-dependent protein kinase I (CaMKI), and a kinaseactivity of BDNF/NT-3 growth factors receptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2), a kinase activity of calcium/calmodulin-dependentprotein kinase I (CaMKI), and a kinase activity of BDNF/NT-3 growthfactors receptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 2 (MK2).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 3 (MK3).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of calcium/calmodulin-dependentprotein kinase I (CaMKI).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of BDNF/NT-3 growth factors receptor(TrkB).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 2 (MK2) and at least 65% of the kinaseactivity of Mitogen-Activated Protein Kinase-Activated Protein Kinase 3(MK3).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 2 (MK2) and at least 65% of the kinaseactivity of calcium/calmodulin-dependent protein kinase I (CaMKI).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 2 (MK2) and at least 65% of the kinaseactivity of BDNF/NT-3 growth factors receptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 2 (MK2), at least 65% of the kinaseactivity of Mitogen-Activated Protein Kinase-Activated Protein Kinase 3(MK3), at least 65% of the kinase activity ofcalcium/calmodulin-dependent protein kinase I (CaMKI), and at least 65%of the kinase activity of BDNF/NT-3 growth factors receptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsthe kinase activity of at least one kinase selected from the group ofMK2, MK3, CaMKI, TrkB, without substantially inhibiting the activity ofone or more other selected kinases from the remaining group listed inTable 1 herein.

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of a kinase selected from the group listed in Table 1herein.

According to another embodiment, this inhibition may, for example, beeffective to reduce fibroblast proliferation, extracellular matrixdeposition, or a combination thereof in the tissue of the subject.

According to another embodiment, this inhibition may, for example, beeffective to reduce at least one pathology selected from the groupconsisting of an aberrant deposition of an extracellular matrix proteinin a pulmonary interstitium, an aberrant promotion of fibroblastproliferation in the lung, an aberrant induction of myofibroblastdifferentiation, and an aberrant promotion of attachment ofmyofibroblasts to an extracellular matrix, compared to a normal healthycontrol subject.

According to some embodiments, inhibitory profiles of MMI inhibitors invivo depend on dosages, routes of administration, and cell typesresponding to the inhibitors.

According to such embodiment, the pharmaceutical composition inhibitsless than 50% of the kinase activity of the other selected kinase(s).According to such embodiment, the pharmaceutical composition inhibitsless than 65% of the kinase activity of the other selected kinase(s).According to another embodiment, the pharmaceutical composition inhibitsless than 50% of the kinase activity of the other selected kinase(s).According to another embodiment, the pharmaceutical composition inhibitsless than 40% of the kinase activity of the other selected kinase(s).According to another embodiment, the pharmaceutical composition inhibitsinhibits less than 20% of the kinase activity of the other selectedkinase(s). According to another embodiment, the pharmaceuticalcomposition inhibits less than 15% of the kinase activity of the otherselected kinase(s). According to another embodiment, the pharmaceuticalcomposition inhibits less than 10% of the kinase activity of the otherselected kinase(s). According to another embodiment, the pharmaceuticalcomposition inhibits less than 5% of the kinase activity of the otherselected kinase(s). According to another embodiment, the pharmaceuticalcomposition increases the kinase activity of the other selected kinases.

According to the embodiments of the immediately preceding paragraph, theone or more other selected kinase that is not substantially inhibited isselected from the group of Ca²⁺/calmodulin-dependent protein kinase II(CaMKII, including its subunit CaMKIIδ), Proto-oncogeneserine/threonine-protein kinase (PIM-1), cellular-Sarcoma (c-SRC),Spleen Tyrosine Kinase (SYK), C-src Tyrosine Kinase (CSK), andInsulin-like Growth Factor 1 Receptor (IGF-1R).

According to some embodiments, the pharmaceutical composition furthercomprises an additional therapeutic agent.

According to some such embodiments, the additional therapeutic agent isone or more selected from the group consisting of purified bovine Type Vcollagens (e.g., IW-001; ImmuneWorks; United Therapeutics), IL-13receptor antagonists (e.g., QAX576; Novartis), protein tyrosine kinaseinhibitors (e.g., imatinib (Gleevec®); Craig Daniels/Novartis),endothelial receptor antagonists (e.g., ACT-064992 (macitentan);Actelion), dual endothelin receptor antagonists (e.g., bosentan(Tracleer®); Actelion), prostacyclin analogs (inhaled iloprost (e.g.,Ventavis®); Actelion), anti-CTGF monoclonal antibodies (e.g., FG-3019),endothelin receptor antagonists (A-selective) (e.g., ambrisentan(Letairis®), Gilead), AB0024 (Arresto), lysyl oxidase-like 2 (LOXL2)monoclonal antibodies (e.g., GS-6624 (formerly AB0024); Gilead), c-JunN-terminal kinase (JNK) inhibitors (e.g., CC-930; Celgene), Pirfenidone(e.g., Esbriet® (InterMune), Pirespa® (Shionogi)), IFN-γ1b (e.g.,Actimmune®; InterMune), pan-neutralizing IgG4 human antibodies againstall three TGF-β isoforms (e.g., GC1008; Genzyme), TGF-β activationinhibitors (e.g., Stromedix (STX-100)), recombinant human Pentraxin-2protein (rhPTX-2) (e.g., PRM151; Promedior), bispecific IL4/IL13antibodies (e.g., SAR156597; Sanofi), humanized monoclonal antibodiestargeting integrin αvβ6 (BIBF 1120; Boehringer Ingelheim),N-acetylcysteine (Zambon SpA), Sildenafil (Viagra®), TNF antagonists(e.g., etanercept (Enbrel®); Pfizer), glucocorticoids (e.g., prednisone,budesonide, mometasone furoate, fluticasone propionate, and fluticasonefuroate), bronchodilators (e.g., leukotriene modifers (e.g., Montelukast(SINGUAIR®)), anticholingertic bronchodilators (e.g., Ipratropiumbromide and Tiotropium), short-acting β2-agonists (e.g., isoetharinemesylate (Bronkometer®), adrenalin, salbutanol/albuterol, andterbutaline), long-acting β2-agonists (e.g., salmeterol, formoterol,indecaterol (Onbrez®), sofosbuvir, an HCV boosted protease inhibitor(ABT-450, AbbVie), a nonnucleoside NS5B inhibitor (dasabuvir, ABT-333,AbbVie), an NS5a inhibitor (ombitasvir, ABT-267, AbbVie), ABT-450/r(ABT-450 with ritonavir), ABT-450 co-formulated with ABT-267, or ABT-450formulated with sofosbuvir. According to another embodiment, anadditional therapeutic agent for HCV treatment is ribavirin, and acombination thereof.

According to some other embodiments, the additional therapeutic agentcomprises a bronchodilator including, but not limited to, a leukotrienemodifier, an anticholinergic bronchodilator, a β2-agonist, or acombination thereof.

According to another embodiment, the additional therapeutic agentcomprises a corticosteroid including, but not limited to, prednisone,budesonide, mometasone, beclemethasone, sofosbuvir (Sovaldi®) or acombination thereof.

According to some other embodiments, the additional therapeutic agentcomprises a bronchodilator including, but not limited to, a leukotrienemodifier, an anticholinergic bronchodilator, a β2-agonist, or acombination thereof.

According to another embodiment, the additional therapeutic agentcomprises a corticosteroid including, but not limited to, prednisone,budesonide, mometasone, beclemethasone, or a combination thereof.

According to another embodiment, the additional therapeutic agent is ananti-inflammatory agent.

According to another embodiment, the anti-inflammatory agent is anonsteroidal anti-inflammatory agent. Mixtures of non-steroidalanti-inflammatory agents also may be employed, as well as thedermatologically acceptable salts and esters of these agents. Forexample, etofenamate, a flufenamic acid derivative, is particularlyuseful for topical application.

According to another embodiment, wherein the nonsteroidalanti-inflammatory agent comprises Transforming Growth Factor-β3(TGF-β3), an anti-Tumor Necrosis Factor-alpha (TNF-α) agent, or acombination thereof.

According to another embodiment, the anti-inflammatory agent is asteroidal anti-inflammatory agent. According to another embodiment, thesteroidal anti-inflammatory agent comprises at least one corticosteroidselected from the group consisting of prednisone, budesonide,mometasone, beclemethasone, and a combination thereof.

According to another embodiment, the additional therapeutic agentcomprises a methylxanthine.

According to another embodiment, the additional therapeutic agentcomprises a neutrophil elastase inhibitor.

According to another embodiment, the additional therapeutic agent is atleast one neutrophil elastase inhibitor, including, but not limited to,ICI 200355, ONO-5046, MR-889, L-694,458, CE-1037, GW-311616, TEI-8362,ONO-6818, AE-3763, FK-706, ICI-200,880, ZD-0892, ZD-8321, and acombination thereof.

According to another embodiment, the additional therapeutic agentcomprises at least one phosphodiesterase inhibitor, including, but notlimited to, phosphodiesterase 4 inhibitor. Examples of phosphodiesterase4 inhibitors include, but are not limited to, roflumilast, cilomilast ora combination thereof.

According to another embodiment, the additional therapeutic agent is ananalgesic agent. According to some such embodiments, the analgesic agentis a non-opioid analgesic. According to some other embodiments, theanalgesic is an opioid analgesic.

According to another embodiment, the additional therapeutic agent is ananti-infective agent. According to another embodiment, theanti-infective agent is an antibiotic agent.

According to some embodiments, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) has a substantialsequence identity to amino acid sequence YARAAARQARAKALARQLGVAA (SEQ IDNO: 1).

According to some such embodiments, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) has at least 70percent sequence identity to amino acid sequence YARAAARQARAKALARQLGVAA(SEQ ID NO: 1). According to another embodiment, the functionalequivalent of the polypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) hasat least 80 percent sequence identity to amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1). According to another embodiment,the functional equivalent of the polypeptide YARAAARQARAKALARQLGVAA (SEQID NO: 1) has at least 90 percent sequence identity to amino acidsequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1). According to anotherembodiment, the functional equivalent of the polypeptideYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) has at least 95 percent sequenceidentity to amino acid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence FAKLAARLYRKALARQLGVAA (SEQ ID NO: 3).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence KAFAKLAARLYRKALARQLGVAA (SEQ ID NO: 4).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence YARAAARQARAKALARQLAVA (SEQ ID NO: 5).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence YARAAARQARAKALARQLGVA (SEQ ID NO: 6).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence YARAAARQARAKALNRQLAVAA (SEQ ID NO: 26).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence YARAAARQARAKALNRQLAVA (SEQ ID NO: 27).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence HRRIKAWLKKIKALARQLGVAA (SEQ ID NO: 7).

According to some other embodiments, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is a fusion proteincomprising a first polypeptide operatively linked to a secondpolypeptide, wherein the first polypeptide is of amino acid sequenceYARAAARQARA (SEQ ID NO: 11), and the second polypeptide comprises atherapeutic domain whose sequence has a substantial identity to aminoacid sequence KALARQLGVAA (SEQ ID NO: 2).

According to another embodiment, the second polypeptide has at least 70percent sequence identity to amino acid sequence KALARQLGVAA (SEQ ID NO:2). According to some other embodiments, the second polypeptide has atleast 80 percent sequence identity to amino acid sequence KALARQLGVAA(SEQ ID NO: 2). According to some other embodiments, the secondpolypeptide has at least 90 percent sequence identity to amino acidsequence KALARQLGVAA (SEQ ID NO: 2). According to some otherembodiments, the second polypeptide has at least 95 percent sequenceidentity to amino acid sequence KALARQLGVAA (SEQ ID NO: 2).

According to another embodiment, the second polypeptide is a polypeptideof amino acid sequence KALARQLAVA (SEQ ID NO: 8).

According to another embodiment, the second polypeptide is a polypeptideof amino acid sequence KALARQLGVA (SEQ ID NO: 9).

According to another embodiment, the second polypeptide is a polypeptideof amino acid sequence KALARQLGVAA (SEQ ID NO: 10).

According to another embodiment, the second polypeptide is a polypeptideof amino acid sequence KALNRQLAVAA (SEQ ID NO: 28).

According to another embodiment, the second polypeptide is a polypeptideof amino acid sequence KALNRQLAVA (SEQ ID NO: 29).

According to some embodiments, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is a fusion proteincomprising a first polypeptide operatively linked to a secondpolypeptide, wherein the first polypeptide comprises a cell penetratingpeptide functionally equivalent to YARAAARQARA (SEQ ID NO: 11), and thesecond polypeptide is of amino acid sequence KALARQLGVAA (SEQ ID NO: 2),and the pharmaceutical composition inhibits both the kinase activity ofMitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2).

According to another embodiment, the first polypeptide is a polypeptideof amino acid sequence WLRRIKAWLRRIKA (SEQ ID NO: 12).

According to another embodiment, the first polypeptide is a polypeptideof amino acid sequence WLRRIKA (SEQ ID NO: 13).

According to another embodiment, the first polypeptide is a polypeptideof amino acid sequence YGRKKRRQRRR (SEQ ID NO: 14).

According to another embodiment, the first polypeptide is a polypeptideof amino acid sequence WLRRIKAWLRRI (SEQ ID NO: 15).

According to another embodiment, the first polypeptide is a polypeptideof amino acid sequence FAKLAARLYR (SEQ ID NO: 16). According to somesuch embodiments, the first polypeptide is a polypeptide of amino acidsequence KAFAKLAARLYR (SEQ ID NO: 17).

According to some such embodiments, the first polypeptide is apolypeptide of amino acid sequence HRRIKAWLKKI (SEQ ID NO: 18).

According to another aspect, the described invention also provides anisolated nucleic acid that encodes a protein sequence with at least 70%amino acid sequence identity to amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1).

According to some such embodiments, the isolated nucleic acid encodes aprotein sequence with at least 80% amino acid sequence identity to aminoacid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1). According to someother embodiments, the isolated nucleic acid encodes a protein sequencewith at least 90% amino acid sequence identity to amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1). According to some otherembodiments, the isolated nucleic acid encodes a protein sequence withat least 95% amino acid sequence identity to amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1).

According to another embodiment, the step of administering may occursystemically either orally, buccally, parenterally, topically, byinhalation, by insufflation, or rectally, or may occur locally by meanssuch as, but not limited to, injection, implantation, grafting, topicalapplication, or parenterally. Additional administration may beperformed, for example, intravenously, transmucosally, transdermally,intramuscularly, subcutaneously, intratracheally (including by pulmonaryinhalation), intraperitoneally, intrathecally, intralymphatically,intralesionally, or epidurally. Administering can be performed, forexample, once, a plurality of times, and/or over one or more extendedperiods either as individual unit doses or in the form of a treatmentregimen comprising multiple unit doses of multiple drugs and/orsubstances.

According to some other embodiments, the step of administering occurs atone time as a single dose. According to some other embodiments, the stepof administering is performed as a plurality of doses over a period oftime. According to some such embodiments, the period of time is a day, aweek, a month, a month, a year, or multiples thereof. According to someembodiments, the step of administering is performed daily for a periodof at least one week. According to some embodiments, the step ofadministering is performed weekly for a period of at least one month.According to some embodiments, the step of administering is performedmonthly for a period of at least two months. According to anotherembodiment, the step of administering is performed repeatedly over aperiod of at least one year. According to another embodiment, the stepof administering is performed at least once monthly. According toanother embodiment, the step of administering is performed at least onceweekly. According to another embodiment, the step of administering isperformed at least once daily.

According to some other embodiments, the therapeutic amount of thepharmaceutical composition is administered via an inhalation device.Examples of the inhalation device that can be used for administering thepharmaceutical composition include, but are not limited to, a nebulizer,a metered-dose inhaler (MDI), a dry powder inhaler (DPI), and a drypowder nebulizer.

According to another embodiment, the dry powder comprises microparticleswith Mass Median Aerodynamic Diameter (MMAD) of 1 to 5 microns.According to another embodiment, the dry powder comprises microparticleswith Mass Median Aerodynamic Diameter (MMAD) of about 2 micron.

According to some other embodiments, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 100 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.00001 mg/kg body weight to about 100 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.0001 mg/kg body weight to about 100 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.001 mg/kg body weight to about 10 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.01 mg/kg body weight to about 10 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.1 mg/kg (or 100 μg/kg) body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 1 mg/kg body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 10 mg/kg body weight to about 100mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 2 mg/kg body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 3 mg/kg body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 4 mg/kg body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 5 mg/kg body weight to about 10mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 60 mg/kg body weight to about 100mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 70 mg/kg body weight to about 100mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 80 mg/kg body weight to about 100mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitory peptide of the pharmaceuticalcomposition is of an amount from about 90 mg/kg body weight to about 100mg/kg body weight. According to another embodiment, the therapeuticamount of the therapeutic inhibitor peptide of the pharmaceuticalcomposition is of an amount from about 0.000001 mg/kg body weight toabout 90 mg/kg body weight. According to another embodiment, thetherapeutic amount of the therapeutic inhibitor peptide of thepharmaceutical composition is of an amount from about 0.000001 mg/kgbody weight to about 80 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 70 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 60 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 50 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 40 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideis of an amount from about 0.000001 mg/kg body weight to about 30 mg/kgbody weight. According to another embodiment, the therapeutic amount ofthe therapeutic inhibitor peptide of the pharmaceutical composition isof an amount from about 0.000001 mg/kg body weight to about 20 mg/kgbody weight. According to another embodiment, the therapeutic amount ofthe therapeutic inhibitor peptide of the pharmaceutical composition isof an amount from about 0.000001 mg/kg body weight to about 10 mg/kgbody weight. According to another embodiment, the therapeutic amount ofthe therapeutic inhibitor peptide of the pharmaceutical composition isof an amount from about 0.000001 mg/kg body weight to about 1 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.1 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.1 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.01 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.001 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.0001 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 0.00001 mg/kg bodyweight.

According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 1 μg/kg/day to 25 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 1 μg/kg/day to 2 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 2 μg/kg/day to 3 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 3 μg/kg/day to 4 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical ranges from 4μg/kg/day to 5 μg/kg/day. According to some other embodiments, thetherapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 5 μg/kg/day to 6 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 6 μg/kg/day to 7 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 7 μg/kg/day to 8 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 8 μg/kg/day to 9 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 9 μg/kg/day to 10 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 1 μg/kg/day to 5 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 5 μg/kg/day to 10 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 10 μg/kg/day to 15 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 15 μg/kg/day to 20 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 25 μg/kg/day to 30 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 30 μg/kg/day to 35 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 35 μg/kg/day to 40 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 40 μg/kg/day to 45 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 45 μg/kg/day to 50 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 50 μg/kg/day to 55 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 55 μg/kg/day to 60 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 60 μg/kg/day to 65 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 65 μg/kg/day to 70 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 70 μg/kg/day to 75 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 80 μg/kg/day to 85 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 85 μg/kg/day to 90 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 90 μg/kg/day to 95 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 95 μg/kg/day to 100 μg/kg/day.

According to another embodiment, the therapeutic dose of the therapeuticinhibitor peptide of the pharmaceutical composition is 1 μg/kg/day.

According to another embodiment, the therapeutic dose of the therapeuticinhibitor peptide of the pharmaceutical composition is 2 μg/kg/day.

According to another embodiment, the therapeutic dose of the therapeuticinhibitor peptide of the pharmaceutical composition is 5 μg/kg/day.

According to another embodiment, the therapeutic dose of the therapeuticinhibitor peptide of the pharmaceutical composition is 10 μg/kg/day.

III. Systems for Preventing or Treating Diseases Characterized byAberrant Fibroblast Proliferation and Collagen Deposition

According to another aspect, the described invention provides a systemfor the treatment of a disease, condition, or process characterized byaberrant fibroblast proliferation and extracellular matrix deposition ina tissue of a subject,

wherein the pharmaceutical composition comprises a therapeutic amount ofa polypeptide of the amino acid sequence YARAAARQARAKALARQLGVAA (SEQ IDNO: 1) or a functional equivalent thereof, and a pharmaceuticallyacceptable carrier thereof, and wherein the therapeutic amount iseffective to reduce the fibroblast proliferation and extracellularmatrix deposition in the tissue of the subject.

According to one embodiment of the method, the disease or the conditionis Acute Lung Injury (ALI) or acute respiratory distress syndrome(ARDS).

According to another embodiment, the disease or the condition isradiation-induced fibrosis.

According to another embodiment, the disease or the condition istransplant rejection.

According to another embodiment, the tissue is a lung tissue.

According to another embodiment, the disease or the condition is aninterstitial lung disease.

According to another embodiment, the disease or the condition ispulmonary fibrosis.

According to another embodiment, the pulmonary fibrosis is idiopathicpulmonary fibrosis.

According to another embodiment, the pulmonary fibrosis results fromadministration of bleomycin.

According to another embodiment, the pulmonary fibrosis results from anallergic reaction, inhalation of environmental particulates, a bacterialinfection, a viral infection, mechanical damage to a lung of thesubject, lung transplantation rejection, an autoimmune disorder, agenetic disorder, or a combination thereof.

According to another embodiment, the tissue is a liver tissue.

According to another embodiment, the disease or condition is liverfibrosis.

According to another embodiment, the tissue is a kidney tissue.

According to another embodiment, the disease or condition is renalfibrosis.

According to another embodiment, the tissue is vascular tissue.

According to another embodiment, the disease or condition is vascularfibrosis.

According to another embodiment, the disease or condition is furthercharacterized by an inflammation in the tissue.

According to another embodiment, the inflammation is an acute or achronic inflammation.

According to another embodiment, the inflammation is mediated by atleast one cytokine selected from the group consisting of Tumor NecrosisFactor-alpha (TNF-α), Interleukin-6 (IL-6), and Interleukin-1β (IL-1β).

According to another embodiment, the aberrant fibroblast proliferationand extracellular matrix deposition in the tissue is characterized by anaberrant activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2) in the tissue compared to the activity ofMitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2) in thetissue of a normal healthy control subject.

According to another embodiment, the aberrant fibroblast proliferationand extracellular matrix deposition in the tissue is evidenced by anaberrant amount or distribution of activated (phosphorylated)Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2) in thetissue compared to the amount or distribution of activatedMitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2) in thetissue of a normal healthy control subject.

According to another embodiment, the pulmonary fibrosis is characterizedby at least one pathology selected from the group consisting of anaberrant deposition of an extracellular matrix protein in a pulmonaryinterstitium, an aberrant promotion of fibroblast proliferation in thelung, an aberrant induction of differentiation of a population offibroblasts into a population of myofibroblasts in the lung, and anaberrant promotion of attachment of myofibroblasts to an extracellularmatrix compared to a normal healthy control subject.

According to another embodiment, the pharmaceutically acceptable carrierincludes, but is not limited to, a controlled release carrier, a delayedrelease carrier, a sustained release carrier, and a long-term releasecarrier.

According to another embodiment, the inhalation device is a nebulizer.

According to another embodiment, the inhalation device is a metered-doseinhaler (MDI).

According to another embodiment, the inhalation device is a dry powderinhaler (DPI).

According to another embodiment, the inhalation device is a dry powdernebulizer.

According to another embodiment, the pharmaceutical composition is in aform of a dry powder.

According to another embodiment, the dry powder comprises microparticleswith Mass Median Aerodynamic Diameter (MMAD) of 1 to 5 microns.

According to another embodiment, the dry powder comprises microparticleswith Mass Median Aerodynamic Diameter (MMAD) of about 2 micron.

According to some embodiments, the pharmaceutical composition furthercomprises an additional therapeutic agent.

According to some such embodiments, the additional therapeutic agent isselected from the group consisting of purified bovine Type V collagens(e.g., IW-001; ImmuneWorks; United Therapeutics), IL-13 receptorantagonists (e.g., QAX576; Novartis), protein tyrosine kinase inhibitors(e.g., imatinib (Gleevec®); Craig Daniels/Novartis), endothelialreceptor antagonists (e.g., ACT-064992 (macitentan); Actelion), dualendothelin receptor antagonists (e.g., bosentan (Tracleer®); Actelion),prostacyclin analogs (inhaled iloprost (e.g., Ventavis®); Actelion),anti-CTGF monoclonal antibodies (e.g., FG-3019), endothelin receptorantagonists (A-selective) (e.g., ambrisentan (Letairis®), Gilead),AB0024 (Arresto), lysyl oxidase-like 2 (LOXL2) monoclonal antibodies(e.g., GS-6624 (formerly AB0024); Gilead), c-Jun N-terminal kinase (JNK)inhibitors (e.g., CC-930; Celgene), Pirfenidone (e.g., Esbriet®(InterMune), Pirespa® (Shionogi)), IFN-γ1b (e.g., Actimmune®;InterMune), pan-neutralizing IgG4 human antibodies against all threeTGF-β isoforms (e.g., GC1008; Genzyme), TGF-β activation inhibitors(e.g., Stromedix (STX-100)), recombinant human Pentraxin-2 protein(rhPTX-2) (e.g., PRM151; Promedior), bispecific IL4/IL13 antibodies(e.g., SAR156597; Sanofi), humanized monoclonal antibodies targetingintegrin αvβ6 (BIBF 1120; Boehringer Ingelheim), N-acetylcysteine(Zambon SpA), Sildenafil (Viagra®), TNF antagonists (e.g., etanercept(Enbrel®); Pfizer), glucocorticoids (e.g., prednisone, budesonide,mometasone furoate, fluticasone propionate, and fluticasone furoate),bronchodilators (e.g., leukotriene modifers (e.g., Montelukast(SINGUAIR®)), anticholingertic bronchodilators (e.g., Ipratropiumbromide and Tiotropium), short-acting β2-agonists (e.g., isoetharinemesylate (Bronkometer®), adrenalin, salbutanol/albuterol, andterbutaline), long-acting β2-agonists (e.g., salmeterol, formoterol,indecaterol (Onbrez®), sofosbuvir (Sovaldi®), an HCV boosted proteaseinhibitor (ABT-450, AbbVie), a nonnucleoside NS5B inhibitor (dasabuvir,ABT-333, AbbVie), an NS5a inhibitor (ombitasvir, ABT-267, AbbVie),ABT-450/r (ABT-450 with ritonavir), ABT-450 co-formulated with ABT-267,ABT-450 formulated with sofosbuvir, ribavirin, and combinations thereof.

According to some other embodiments, the additional therapeutic agentcomprises a bronchodilator including, but not limited to, a leukotrienemodifier, an anticholinergic bronchodilator, a β2-agonist, or acombination thereof.

According to another embodiment, the additional therapeutic agentcomprises a corticosteroid including, but not limited to, prednisone,budesonide, mometasone, beclemethasone, or a combination thereof.

According to some such embodiments, the additional therapeutic agentcomprises a bronchodilator including, but not limited to, a leukotrienemodifier, an anticholinergic bronchodilator, a β2-agonist, or acombination thereof.

According to another embodiment, the additional therapeutic agentcomprises a corticosteroid including, but not limited to, prednisone,budesonide, mometasone, beclemethasone, or a combination thereof.

According to another embodiment, the additional therapeutic agent is ananti-inflammatory agent.

According to another embodiment, the anti-inflammatory agent is anonsteroidal anti-inflammatory agent. Mixtures of non-steroidalanti-inflammatory agents also may be employed, as well as thedermatologically acceptable salts and esters of these agents. Forexample, etofenamate, a flufenamic acid derivative, is particularlyuseful for topical application.

According to another embodiment, wherein the nonsteroidalanti-inflammatory agent comprises Transforming Growth Factor-β3(TGF-β3), an anti-Tumor Necrosis Factor-alpha (TNF-α) agent, or acombination thereof.

According to another embodiment, the anti-inflammatory agent is asteroidal anti-inflammatory agent. According to another embodiment, thesteroidal anti-inflammatory agent comprises at least one corticosteroidselected from the group consisting of prednisone, budesonide,mometasone, beclemethasone, and a combination thereof.

According to another embodiment, the additional therapeutic agentcomprises a methylxanthine.

According to another embodiment, the additional therapeutic agentcomprises a neutrophil elastase inhibitor.

According to another embodiment, the additional therapeutic agent is atleast one neutrophil elastase inhibitor, including, but not limited to,ICI 200355, ONO-5046, MR-889, L-694,458, CE-1037, GW-311616, TEI-8362,ONO-6818, AE-3763, FK-706, ICI-200,880, ZD-0892, ZD-8321, and acombination thereof.

According to another embodiment, the additional therapeutic agentcomprises at least one phosphodiesterase inhibitor, including, but notlimited to, phosphodiesterase 4 inhibitor. Examples of phosphodiesterase4 inhibitors include, but are not limited to, roflumilast, cilomilast ora combination thereof.

According to another embodiment, the additional therapeutic agent is ananalgesic agent. According to some such embodiments, the analgesic agentis a non-opioid analgesic. According to some other embodiments, theanalgesic is an opioid analgesic.

According to another embodiment, the additional therapeutic agent is ananti-infective agent. According to another embodiment, theanti-infective agent is an antibiotic agent.

According to another embodiment, the additional therapeutic agent is ananti-viral agent. Examplary such antiviral agents include, withoutlimitation, an HCV boosted protease inhibitor (ABT-450, AbbVie), anonnucleoside NS5B inhibitor (dasabuvir, ABT-333, AbbVie), an NS5ainhibitor (ombitasvir, ABT-267, AbbVie), ABT-450/r (ABT-450 withritonavir), ABT-450 co-formulated with ABT-267, ABT-450 formulated withsofosbuvir, ribavirin, or sofosbuvir. According to another embodiment,the pharmaceutical composition inhibits inflammation occurring in a lungof the subject. According to another embodiment, the inflammation is anacute inflammation. According to another embodiment, the inflammation isa chronic inflammation. According to another embodiment, theinflammation is mediated by an elevated level of Tumor NecrosisFactor-alpha (TNF-α). According to another embodiment, the inflammationis mediated by an elevated level of Interleukin-6 (IL-6). According toanother embodiment, the inflammation is mediated by an elevated level ofInterleukin-1β (IL-1β).

According to another embodiment, the pharmaceutical compositionmodulates an amount of Tumor Necrosis Factor-alpha (TNF-α) in the lung,compared to a control. According to another embodiment, thepharmaceutical composition modulates an amount of Interleukin-6 (IL-6)in the lung, compared to a control. According to another embodiment, thepharmaceutical composition modulates an amount of Interleukin-1β (IL-1β)in the lung, compared to a control.

According to another embodiment, the pharmaceutical composition inhibitsan activity of HSPB1. According to another embodiment, the activity ofHSPB1 inhibited by the pharmaceutical composition is an aberrantinduction of fibroblast proliferation. According to another embodiment,the activity of HSPB1 inhibited by the pharmaceutical composition is anaberrant induction of differentiation of a population of fibroblastsinto a population of myofibroblasts. According to another embodiment,the activity of HSPB1 inhibited by the pharmaceutical composition is adeposition of an extracellular matrix protein into a pulmonaryinterstitium. According to another embodiment, the extracellular matrixprotein is collagen. According to another embodiment, the activity ofHSPB1 inhibited by the pharmaceutical composition is a promotion offibrotic loci formation. According to another embodiment, the activityof HSPB1 inhibited by the pharmaceutical composition is an increase ofmyofibroblast contractile activity. According to another embodiment, theactivity of HSPB1 inhibited by the pharmaceutical composition is apromotion of myofibroblast attachment to extracellular matrix.

According to another embodiment, the aberrant fibroblast proliferationand extracellular matrix deposition in the tissue is evidenced by anaberrant amount or distribution of activated (phosphorylated)Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2) in thetissue compared to the amount or distribution of activatedMitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2) in thetissue of a normal healthy control subject.

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of a kinase selected from the group listed in Table 1herein.

According to another embodiment, the pharmaceutical composition inhibitsat least 50% of the kinase activity of the kinase. According to anotherembodiment, the pharmaceutical composition inhibits at least 65% of thekinase activity of the kinase. According to another embodiment, thepharmaceutical composition inhibits at least 75% of the kinase activityof that kinase. According to another embodiment, the pharmaceuticalcomposition inhibits at least 80% of the kinase activity of that kinase.According to another embodiment, the pharmaceutical composition inhibitsat least 85% of the kinase activity of that kinase. According to anotherembodiment, the pharmaceutical composition inhibits at least 90% of thekinase activity of that kinase. According to another embodiment, thepharmaceutical composition inhibits at least 95% of the kinase activityof that kinase.

According to some embodiments, inhibitory profiles of MMI inhibitors invivo depend on dosages, routes of administration, and cell typesresponding to the inhibitors.

According to another embodiment, the pharmaceutical composition inhibitsat least 50% of the kinase activity of the kinase. According to anotherembodiment, the pharmaceutical composition inhibits at least 65% of thekinase activity of the kinase. According to another embodiment, thepharmaceutical composition inhibits at least 75% of the kinase activityof that kinase. According to another embodiment, the pharmaceuticalcomposition inhibits at least 80% of the kinase activity of that kinase.According to another embodiment, the pharmaceutical composition inhibitsat least 85% of the kinase activity of that kinase. According to anotherembodiment, the pharmaceutical composition inhibits at least 90% of thekinase activity of that kinase. According to another embodiment, thepharmaceutical composition inhibits at least 95% of the kinase activityof that kinase.

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2 kinase). According to another embodiment, thepharmaceutical composition inhibits at least 50% of the kinase activityof MK2 kinase. According to another embodiment, the pharmaceuticalcomposition inhibits at least 65% of the kinase activity of MK2 kinase.According to another embodiment, the pharmaceutical composition inhibitsat least 75% of the kinase activity of MK2 kinase. According to anotherembodiment, the pharmaceutical composition inhibits at least 80% of thekinase activity of MK2 kinase. According to another embodiment, thepharmaceutical composition inhibits at least 85% of the kinase activityof MK2 kinase. According to another embodiment, the pharmaceuticalcomposition inhibits at least 90% of the kinase activity of MK2 kinase.According to another embodiment, the pharmaceutical composition inhibitsat least 95% of the kinase activity of MK2 kinase.

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 3 (MK3 kinase). According to another embodiment, thepharmaceutical composition further inhibits at least 50% of the kinaseactivity of MK3 kinase. According to another embodiment, thepharmaceutical composition further inhibits at least 65% of the kinaseactivity of MK3 kinase. According to another embodiment, thepharmaceutical composition further inhibits at least 70% of the kinaseactivity of MK3 kinase. According to another embodiment, thepharmaceutical composition further inhibits at least 75% of the kinaseactivity of MK3 kinase. According to another embodiment, thepharmaceutical composition further inhibits at least 80% of the kinaseactivity of MK3 kinase. According to another embodiment, thepharmaceutical composition further inhibits at least 85% of the kinaseactivity of MK3 kinase. According to another embodiment, thepharmaceutical composition further inhibits at least 90% of the kinaseactivity of MK3 kinase. According to another embodiment, thepharmaceutical composition further inhibits at least 95% of the kinaseactivity of MK3 kinase.

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of calcium/calmodulin-dependent protein kinase I(CaMKI). According to another embodiment, the pharmaceutical compositionfurther inhibits at least 50% of the kinase activity ofCa²⁺/calmodulin-dependent protein kinase I (CaMKI). According to anotherembodiment, the pharmaceutical composition further inhibits at least 65%of the kinase activity of Ca²⁺/calmodulin-dependent protein kinase I(CaMKI). According to another embodiment, the pharmaceutical compositionfurther inhibits at least 70% of the kinase activity ofCa²⁺/calmodulin-dependent protein kinase I (CaMKI). According to anotherembodiment, the pharmaceutical composition further inhibits at least 75%of the kinase activity of Ca²⁺/calmodulin-dependent protein kinase I(CaMKI). According to another embodiment, the pharmaceutical compositionfurther inhibits at least 80% of the kinase activity ofCa²⁺/calmodulin-dependent protein kinase I (CaMKI). According to anotherembodiment, the pharmaceutical composition further inhibits at least 85%of the kinase activity of Ca²⁺/calmodulin-dependent protein kinase I(CaMKI). According to another embodiment, the pharmaceutical compositionfurther inhibits at least 90% of the kinase activity ofCa²⁺/calmodulin-dependent protein kinase I (CaMKI). According to anotherembodiment, the pharmaceutical composition further inhibits at least 95%of the kinase activity of Ca²⁺/calmodulin-dependent protein kinase I(CaMKI).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of BDNF/NT-3 growth factors receptor (TrkB). Accordingto another embodiment, the pharmaceutical further inhibits at least 50%of the kinase activity of BDNF/NT-3 growth factors receptor (TrkB).According to another embodiment, the pharmaceutical further inhibits atleast 65% of the kinase activity of BDNF/NT-3 growth factors receptor(TrkB). According to another embodiment, the pharmaceutical furtherinhibits at least 70% of the kinase activity of BDNF/NT-3 growth factorsreceptor (TrkB). According to another embodiment, the pharmaceuticalfurther inhibits at least 75% of the kinase activity of BDNF/NT-3 growthfactors receptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2) and a kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 3 (MK3).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2) and a kinase activity of calcium/calmodulin-dependentprotein kinase I (CaMKI).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2) and a kinase activity of BDNF/NT-3 growth factorsreceptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2), a kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 3 (MK3), a kinase activity ofcalcium/calmodulin-dependent protein kinase I (CaMKI), and a kinaseactivity of BDNF/NT-3 growth factors receptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of Mitogen-Activated Protein Kinase-Activated ProteinKinase 2 (MK2), a kinase activity of calcium/calmodulin-dependentprotein kinase I (CaMKI), and a kinase activity of BDNF/NT-3 growthfactors receptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 2 (MK2).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 3 (MK3).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of calcium/calmodulin-dependentprotein kinase I (CaMKI).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of BDNF/NT-3 growth factors receptor(TrkB).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 2 (MK2) and at least 65% of the kinaseactivity of Mitogen-Activated Protein Kinase-Activated Protein Kinase 3(MK3).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 2 (MK2) and at least 65% of the kinaseactivity of calcium/calmodulin-dependent protein kinase I (CaMKI).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 2 (MK2) and at least 65% of the kinaseactivity of BDNF/NT-3 growth factors receptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsat least 65% of the kinase activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 2 (MK2), at least 65% of the kinaseactivity of Mitogen-Activated Protein Kinase-Activated Protein Kinase 3(MK3), at least 65% of the kinase activity ofcalcium/calmodulin-dependent protein kinase I (CaMKI), and at least 65%of the kinase activity of BDNF/NT-3 growth factors receptor (TrkB).

According to another embodiment, the pharmaceutical composition inhibitsthe kinase activity of at least one kinase selected from the group ofMK2, MK3, CaMKI, TrkB, without substantially inhibiting the activity ofone or more other selected kinases from the remaining group listed inTable 1 herein.

According to another embodiment, the pharmaceutical composition inhibitsa kinase activity of a kinase selected from the group listed in Table 1herein.

According to another embodiment, this inhibition may, for example, beeffective to reduce fibroblast proliferation, extracellular matrixdeposition, or a combination thereof in the tissue of the subject.

According to another embodiment, this inhibition may, for example, beeffective to reduce at least one pathology selected from the groupconsisting of an aberrant deposition of an extracellular matrix proteinin a pulmonary interstitium, an aberrant promotion of fibroblastproliferation in the lung, an aberrant induction of myofibroblastdifferentiation, and an aberrant promotion of attachment ofmyofibroblasts to an extracellular matrix, compared to a normal healthycontrol subject.

According to some embodiments, inhibitory profiles of MMI inhibitors invivo depend on dosages, routes of administration, and cell typesresponding to the inhibitors.

According to such embodiment, the pharmaceutical composition inhibitsless than 50% of the kinase activity of the other selected kinase(s).According to such embodiment, the pharmaceutical composition inhibitsless than 65% of the kinase activity of the other selected kinase(s).According to such embodiment, the pharmaceutical composition inhibitsless than 50% of the kinase activity of the other selected kinase(s).According to another embodiment, the pharmaceutical composition inhibitsless than 40% of the kinase activity of the other selected kinase(s).According to another embodiment, the pharmaceutical composition inhibitsinhibits less than 20% of the kinase activity of the other selectedkinase(s). According to another embodiment, the pharmaceuticalcomposition inhibits less than 15% of the kinase activity of the otherselected kinase(s). According to another embodiment, the pharmaceuticalcomposition inhibits less than 10% of the kinase activity of the otherselected kinase(s). According to another embodiment, the pharmaceuticalcomposition inhibits less than 5% of the kinase activity of the otherselected kinase(s). According to another embodiment, the pharmaceuticalcomposition increases the kinase activity of the other selected kinases.

According to the embodiments of the immediately preceding paragraph, theone or more other selected kinase that is not substantially inhibited isselected from the group of Ca²⁺/calmodulin-dependent protein kinase II(CaMKII, including its subunit CaMKIIδ), Proto-oncogeneserine/threonine-protein kinase (PIM-1), cellular-Sarcoma (c-SRC),Spleen Tyrosine Kinase (SYK), C-src Tyrosine Kinase (CSK), andInsulin-like Growth Factor 1 Receptor (IGF-1R).

According to some embodiments, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) has a substantialsequence identity to amino acid sequence YARAAARQARAKALARQLGVAA (SEQ IDNO: 1).

According to another embodiments, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) has at least 70percent sequence identity to amino acid sequence YARAAARQARAKALARQLGVAA(SEQ ID NO: 1). According to another embodiment, the functionalequivalent of the polypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) hasat least 80 percent sequence identity to amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1). According to another embodiment,the functional equivalent of the polypeptide YARAAARQARAKALARQLGVAA (SEQID NO: 1) has at least 90 percent sequence identity to amino acidsequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1). According to anotherembodiment, the functional equivalent of the polypeptideYARAAARQARAKALARQLGVAA (SEQ ID NO: 1) has at least 95 percent sequenceidentity to amino acid sequence YARAAARQARAKALARQLGVAA (SEQ ID NO: 1).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence FAKLAARLYRKALARQLGVAA (SEQ ID NO: 3).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence KAFAKLAARLYRKALARQLGVAA (SEQ ID NO: 4).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence YARAAARQARAKALARQLAVA (SEQ ID NO: 5).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence YARAAARQARAKALARQLGVA (SEQ ID NO: 6).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence HRRIKAWLKKIKALARQLGVAA (SEQ ID NO: 7).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence YARAAARQARAKALNRQLAVAA (SEQ ID NO: 26).

According to another embodiment, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is of amino acidsequence YARAAARQARAKALNRQLAVA (SEQ ID NO: 27).

According to some other embodiments, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is a fusion proteincomprising a first polypeptide operatively linked to a secondpolypeptide, wherein the first polypeptide is of amino acid sequenceYARAAARQARA (SEQ ID NO: 11), and the second polypeptide comprises atherapeutic domain whose sequence has a substantial identity to aminoacid sequence KALARQLGVAA (SEQ ID NO: 2).

According to another embodiment, the second polypeptide has at least 70percent sequence identity to amino acid sequence KALARQLGVAA (SEQ ID NO:2). According to some other embodiments, the second polypeptide has atleast 80 percent sequence identity to amino acid sequence KALARQLGVAA(SEQ ID NO: 2). According to some other embodiments, the secondpolypeptide has at least 90 percent sequence identity to amino acidsequence KALARQLGVAA (SEQ ID NO: 2). According to some otherembodiments, the second polypeptide has at least 95 percent sequenceidentity to amino acid sequence KALARQLGVAA (SEQ ID NO: 2).

According to another embodiment, the second polypeptide is a polypeptideof amino acid sequence KALARQLAVA (SEQ ID NO: 8).

According to another embodiment, the second polypeptide is a polypeptideof amino acid sequence KALARQLGVA (SEQ ID NO: 9).

According to another embodiment, the second polypeptide is a polypeptideof amino acid sequence KALARQLGVAA (SEQ ID NO: 10).

According to another embodiment, the second polypeptide is a polypeptideof amino acid sequence KALNRQLAVAA SEQ ID NO: 28).

According to another embodiment, the second polypeptide is a polypeptideof amino acid sequence KALNRQLAVA SEQ ID NO: 29).

According to some other embodiments, the functional equivalent of thepolypeptide YARAAARQARAKALARQLGVAA (SEQ ID NO: 1) is a fusion proteincomprising a first polypeptide operatively linked to a secondpolypeptide, wherein the first polypeptide comprises a cell penetratingpeptide functionally equivalent to YARAAARQARA (SEQ ID NO: 11), and thesecond polypeptide is of amino acid sequence KALARQLGVAA (SEQ ID NO: 2).

According to a further embodiment, the first polypeptide is apolypeptide of amino acid sequence WLRRIKAWLRRIKA (SEQ ID NO: 12).

According to another embodiment, the first polypeptide is a polypeptideof amino acid sequence WLRRIKA (SEQ ID NO: 13).

According to another embodiment, the first polypeptide is a polypeptideof amino acid sequence YGRKKRRQRRR (SEQ ID NO: 14).

According to another embodiment, the first polypeptide is a polypeptideof amino acid sequence WLRRIKAWLRRI (SEQ ID NO: 15).

According to another embodiment, the first polypeptide is a polypeptideof amino acid sequence FAKLAARLYR (SEQ ID NO: 16).

According to another embodiment, the first polypeptide is a polypeptideof amino acid sequence KAFAKLAARLYR (SEQ ID NO: 17).

According to another embodiment, the first polypeptide is a polypeptideof amino acid sequence HRRIKAWLKKI (SEQ ID NO: 18).

According to another aspect, the described invention also provides anisolated nucleic acid that encodes a protein sequence with at least 70%amino acid sequence identity to amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1). According to some suchembodiments, the isolated nucleic acid encodes a protein sequence withat least 80% amino acid sequence identity to amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1). According to some suchembodiments, the isolated nucleic acid encodes a protein sequence withat least 90% amino acid sequence identity to amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1). According to some suchembodiments, the isolated nucleic acid encodes a protein sequence withat least 95% amino acid sequence identity to amino acid sequenceYARAAARQARAKALARQLGVAA (SEQ ID NO: 1).

According to some other embodiments, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 100 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.00001 mg/kg body weight to about 100 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.0001 mg/kg body weight to about 100 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.001 mg/kg body weight to about 10 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.01 mg/kg body weight to about 10 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 0.1 mg/kg (100 μg/kg) body weight to about 10 mg/kgbody weight. According to another embodiment, the therapeutic amount ofthe therapeutic inhibitory peptide of the pharmaceutical composition isof an amount from about 1 mg/kg body weight to about 10 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 10 mg/kg body weight to about 100 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 2 mg/kg body weight to about 10 mg/kg body weight.According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 3 mg/kg body weight to about 10 mg/kg body weight.According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 4 mg/kg body weight to about 10 mg/kg body weight.According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 5 mg/kg body weight to about 10 mg/kg body weight.According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 60 mg/kg body weight to about 100 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 70 mg/kg body weight to about 100 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 80 mg/kg body weight to about 100 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitory peptide of the pharmaceutical composition is ofan amount from about 90 mg/kg body weight to about 100 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 90 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 80 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 70 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 60 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 50 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide of the pharmaceutical composition is of anamount from about 0.000001 mg/kg body weight to about 40 mg/kg bodyweight. According to another embodiment, the therapeutic amount of thetherapeutic inhibitor peptide is of an amount from about 0.000001 mg/kgbody weight to about 30 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 20 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 10 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 1 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 0.1 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 0.1 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 0.01 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 0.001 mg/kg body weight. According to anotherembodiment, the therapeutic amount of the therapeutic inhibitor peptideof the pharmaceutical composition is of an amount from about 0.000001mg/kg body weight to about 0.0001 mg/kg body weight. According toanother embodiment, the therapeutic amount of the therapeutic inhibitorpeptide of the pharmaceutical composition is of an amount from about0.000001 mg/kg body weight to about 0.00001 mg/kg body weight.

According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 1 μg/kg/day to 25 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 1 μg/kg/day to 2 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 2 μg/kg/day to 3 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 3 μg/kg/day to 4 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical ranges from 4μg/kg/day to 5 μg/kg/day. According to some other embodiments, thetherapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 5 μg/kg/day to 6 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 6 μg/kg/day to 7 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 7 μg/kg/day to 8 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 8 μg/kg/day to 9 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 9 μg/kg/day to 10 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 1 μg/kg/day to 5 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 5 μg/kg/day to 10 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 10 μg/kg/day to 15 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 15 μg/kg/day to 20 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 25 μg/kg/day to 30 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 30 μg/kg/day to 35 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 35 μg/kg/day to 40 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 40 μg/kg/day to 45 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 45 μg/kg/day to 50 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 50 μg/kg/day to 55 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 55 μg/kg/day to 60 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 60 μg/kg/day to 65 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 65 μg/kg/day to 70 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 70 μg/kg/day to 75 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 80 μg/kg/day to 85 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 85 μg/kg/day to 90 μg/kg/day.According to some other embodiments, the therapeutic dose of thetherapeutic inhibitor peptide of the pharmaceutical composition rangesfrom 90 μg/kg/day to 95 μg/kg/day. According to some other embodiments,the therapeutic dose of the therapeutic inhibitor peptide of thepharmaceutical composition ranges from 95 μg/kg/day to 100 μg/kg/day.

According to another embodiment, the therapeutic dose of the therapeuticinhibitor peptide of the pharmaceutical composition is 1 μg/kg/day.

According to another embodiment, the therapeutic dose of the therapeuticinhibitor peptide of the pharmaceutical composition is 2 μg/kg/day.

According to another embodiment, the therapeutic dose of the therapeuticinhibitor peptide of the pharmaceutical composition is 5 μg/kg/day.

According to another embodiment, the therapeutic dose of the therapeuticinhibitor peptide of the pharmaceutical composition is 10 μg/kg/day.

Within this application, unless otherwise stated, the techniquesutilized may be found in any of several well-known references such as:Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, ColdSpring Harbor Laboratory Press), Gene Expression Technology (Methods inEnzymology, Vol. 185, edited by D. Goeddel, 1991. Academic Press, SanDiego, Calif.), “Guide to Protein Purification” in Methods in Enzymology(M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.), Culture of Animal Cells: A Manual of BasicTechnique, 2nd Ed. (R. I. Freshney. 1987. Liss, Inc. New York, N.Y.),and Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J.Murray, The Humana Press Inc., Clifton, N.J.), all of which areincorporated herein by reference.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the described invention, thepreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with thepublications are cited.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range is encompassed within the invention. The upper and lowerlimits of these smaller ranges which may independently be included inthe smaller ranges also is encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits also are included in the invention.

It must also be noted that as used herein and in the appended claims,the singular forms “a,” “and” and “the” include plural referents unlessthe context clearly dictates otherwise. All technical and scientificterms used herein have the same meaning.

The publications discussed herein are incorporated herein by referencein their entirety and are provided solely for their disclosure prior tothe filing date of the present application. Nothing herein is to beconstrued as an admission that the described invention is not entitledto antedate such publication by virtue of prior invention. Further, thedates of publication provided may be different from the actualpublication dates which may need to be independently confirmed.

It should be understood by those skilled in the art that various changesmay be made and equivalents may be substituted without departing fromthe true spirit and scope of the Invention. In addition, manymodifications may be made to adapt a particular situation, material,composition of matter, process, process step or steps, to the objective,spirit and scope of the described invention. All such modifications areintended to be within the scope of the claims appended hereto.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the described invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

I. Materials and Methods MMI-0100 Drug Development

For good manufacturing practice (GMP) production of MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1), approximately 1 kg ofFmoc-Ala-Wang Resin is transferred into a 50 L glass solid phasesynthesis reaction vessel equipped with a mechanical stirrer. The resinis allowed to swell in dimethylformide (DMF) for no less than (NLT) 2hours before draining the DMF. The resin beads then are washed withconsecutive rinses of DMF. The N-terminal protecting group (i.e. Fmoc)is removed (de-blocking step) by treatment with 20% piperidine in DMFand the resin is washed with DMF. The next amino acid in the sequence iscoupled in the presence of 1-hydroxybenzotriazole (HOBt) anddiisopropylcarbodiimide (DIC). Generally, 2.5-3.5 molar equivalents ofFmoc-amino acid (Fmoc-AA) to the synthesis scale are used for coupling.The Fmoc-AA is dissolved in DMF and activated by the addition of HOBtand DIC. The completion of each coupling is monitored by the Ninhydrintest. If a coupling is incomplete, a second coupling with the same aminoacid is performed by using the symmetrical anhydride method. Generally,3.0-6.0 molar equivalents of Fmoc-AA to the synthesis scale are used forcoupling. The Fmoc-AA is dissolved in dichloromethane (DCM) and aminimal volume of DMF and activated through the addition of DIC in amolar ratio of Fmoc-AA/DIC=1.0/0.5. When the full peptide sequence iscompleted, the peptide resin is rinsed thoroughly with successive washesof DMF and MeOH. The resin then is dried under vacuum for NLT 3 hours.Typical recovery of the total dried peptide resin is approximately 2800grams, representing a peptide resin yield of −65%.

Approximately 370-500 grams of peptide resin then are transferred into asuitably sized glass bottle equipped with a magnetic stir bar. The flaskcontaining the peptide resin is cooled in an ice/water bath or in arefrigerator for no later than 30 minutes. The trifluoroacetic acid(TFA) cocktail (a mixture of TFA, TIS, and water in the ratio of 95mL:2.5 mL:2.5 mL) is pre-chilled in an ice/water bath for no later than30 minutes. Approximately 8-12 mL of TFA cleavage cocktail per gram ofresin is added to this vessel. As soon as the peptide resin and TFAcocktail are combined, the ice/water bath is removed and the reactionmixture is stirred at room temperature for 2-3 hours. The reactionmixture then is filtered through a coarse glass filter and the resin iswashed two times with 0.5-1.0 mL of TFA per gram of resin per wash. Thecombined filtrate is collected and the resin is discarded. The filtrateis then added to ether that is pre-chilled in a refrigerator for lessthan 30 minutes, in a ratio of 1 mL of filtrate per 10 mL ether, toprecipitate the cleaved peptide. The peptide-ether mixture isequilibrated to room temperature for no later than 30 minutes. Theprecipitated peptide is collected on a medium glass filter. Theprecipitate is washed thoroughly with cold ether three times, usingenough ether to at least cover all the precipitate on the filter. Theether then is eluted through the same medium glass filter. The crudepeptide is transferred into a plastic bottle and is placed in adesiccator connected to a mechanical vacuum pump to dry for no laterthan 12 hours. After drying, the crude peptide is stored at 5±3° C. Thecleavage procedure is repeated multiple times until all the peptideresin is cleaved. A typical batch recovery of total dried crude peptideis approximately 1250 grams, representing a cleavage yield ofapproximately 110%.

The crude peptide from cleavage is prepared for high-performance liquidchromatography (HPLC) purification by dissolving the peptide in HPLCbuffer at a final crude peptide concentration of 20 mg/mL. The peptidesolution is filtered through a 1 μm glass filter membrane and loadedonto a C18 reverse phase column, which is operated by a preparative HPLCsystem. The column is washed and equilibrated. A linear gradient is usedto elute the crude peptide from the column. Following each crudepurification, the fractions are analyzed by an analytical HPLC systemusing a Kromasil C18, 5 μm, 100 Å 4.6×250 mm column. Fractions generatedfrom the initial purification are pooled based on the HPLC purity andimpurity profile of each fraction. Peptide pools are stored at 2-8° C.until further processing. This process is repeated until all of thecrude peptide was purified through the HPLC column and meet the MainPool purity criteria. A salt exchange to acetate salt is performed byHPLC. The final peptide solution is filtered through a 0.22 μm filterand loaded onto a tray lyophilizer. The peptide is pre-frozen at 40° C.for no later than 720 minutes before starting the lyophilization cycle.The lyophilization takes approximately 5 days. Approximately 50-55%final yield results from the purification and lyophilization steps.

Radiometric IC₅₀ Determination

The IC₅₀ value was estimated from a 10-point curve of one-half logdilutions. Peptide was supplied in dimethyl sulfoxide (DMSO).Specifically, human recombinant MK2 (h) (5-10 mU) was incubated with 50mM sodium 3-glycerophosphate (pH=7.5), 0.1 mM EGTA, 30 μM of substratepeptide (KKLNRTLSVA; SEQ ID NO: 21), 10 mM magnesium acetate, and 90 uMγ-³³P-ATP (final volume of 25 μL) for 40 minutes at room temperature.Then, the reaction was stopped with 3% phosphoric acid. 10 μL of thismixture was spotted onto a P30 filtermat and washed three times for fiveminutes with 75 mM phosphoric acid and once with methanol. Finally, themembrane was dried and a scintillation counter was used. An ATPconcentration within 15 μM of the apparent Km for ATP was chosen,because Hayess and Benndorf (Biochem Pharmacol, 1997, 53(9): 1239-47)showed that the mechanism of their original inhibitor peptide (i.e., thepeptide KKKALNRQLGVAA; SEQ ID NO: 22) was not competitive with ATPbinding.

In addition to determining the IC₅₀ value for MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1), inhibitory activity against 266human kinases was tested using Millipore's IC₅₀ Profiler Express service(Millipore, Billerica, Mass.).

For specificity analysis, 100 μM of each MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1), MMI-0200 (YARAAARQARAKALNRQLGVA;SEQ ID NO: 19), MMI-0300 (FAKLAARLYRKALARQLGVAA; SEQ ID NO: 3), MMI-0400(KAFAKLAARLYRKALARQLGVAA; SEQ ID NO: 4), and MMI-0500(HRRIKAWLKKIKALARQLGVAA; SEQ ID NO: 7), dissolved in dimethyl sulfoxide(DMSO) was used. The 100 μM concentration was chosen because thisconcentration inhibited adhesion formation in an in vivo study (asdisclosed in U.S. application Ser. No. 12/582,516 filed Oct. 20, 2009,the content of which is incorporated herein by reference in itsentirety). Every kinase activity measurement was conducted in duplicate.

Histochemistry and Immunohistochemistry

A mouse model of pulmonary fibrosis was generated by administering 0.025U of bleomycin/PBS intratracheally to C57BL/6 mice. MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or MMI-0200(YARAAARQARAKALNRQLGVA; SEQ ID NO: 19) (at dosages of 50 μg/kg, 75μg/kg, and 100 μg/kg per day) was administered daily starting at day 7post bleomycin injury (for analysis of post-inflammatory/pre-fibroticphase; a prevention model) or at day 14 post bleomycin injury (foranalysis of post-fibrotic phase; treatment model), eitherintraperitoneally or via nebulization, through day 21 or 28 postbleomycin delivery. At 21 day post bleomycin delivery (for preventionmodel) or 28 post bleomycin delivery (for treatment model), groups ofmice were sacrificed with a sodium pentobarbital injection (120 mg/kg)and the chest cavity was opened. The right mainstem bronchus was ligatedand the right lung was removed. The trachea was cannulated and the leftlung was perfused with 4% formaldehyde at 21 cm H₂O pressure. The tissueblocks then were embedded in paraffin, and 4-mm sections were preparedfor staining. Sections from each animal were stained with hematoxylinand eosin (H&E) to visualize cells or with Masson's Trichrome stainingto highlight collagen deposition. After incubation, sections were washedwith 0.2% acetic acid, dehydrated by immersing into 95% alcohol, andcleared with xylene (3-4 times) in a staining dish. Stained sectionswere mounted onto a labeled glass slide with organic mounting medium.

II. Results Example 1 IC₅₀ and Specificity of MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)

IC₅₀ (half maximal inhibitory concentrations) value for the MK2inhibitor peptide (MMI-0100; YARAAARQARAKALARQLGVAA (SEQ ID NO: 1)) wasdetermined using Millipore's IC₅₀ Profiler Express service. Thisquantitative assay measures how much of an inhibitor is needed toinhibit 50% of a given biological process or component of a process(i.e., an enzyme, cell, or cell receptor) [IC₅₀]. Specifically, in theseassays, a positively charged substrate is phosphorylated with aradiolabeled phosphate group from an ATP if the kinase is not inhibitedby an inhibitor peptide. The positively charged substrate then isattracted to a negatively charged filter membrane, quantified with ascintillation counter, and compared to a 100% activity control.

ATP concentrations within 15 μM of the apparent Km for ATP were chosensince an ATP concentration near the Km may allow for the kinases to havethe same relative amount of phosphorylation activity. The IC₅₀ of theMMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) was determined to be 22μM.

In addition to determining the IC₅₀ of the compound, the specificity ofMK2 inhibitory peptides was assessed by examining activities of all 266human kinases available for testing in the Millipore kinase profilingservice (Table 1). For analysis, the kinases that were inhibited morethan 65% by MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1); MMI-0200(YARAAARQARAKALNRQLGVA; SEQ ID NO: 19); MMI-0300 (FAKLAARLYRKALARQLGVAA;SEQ ID NO: 3); MMI-0400 (KAFAKLAARLYRKALARQLGVAA; SEQ ID NO: 4); andMMI-0500 (HRRIKAWLKKIKALARQLGVAA; SEQ ID NO: 7) were determined.

As shown in Table 1, at 100 μM, MK2 inhibitory peptides MMI-0100 (SEQ IDNO: 1), MMI-0200 (SEQ ID NO: 19), MMI-0300 (SEQ ID NO: 3); MMI-0400 (SEQID NO: 4); and MMI-0500 (SEQ ID NO: 5) inhibited a specific group ofkinases and showed very limited off-target kinase inhibition. Morespecifically, MK2 inhibitory peptides MMI-0100 (SEQ ID NO: 1), MMI-0200(SEQ ID NO: 19), MMI-0300 (SEQ ID NO: 3); MMI-0400 (SEQ ID NO: 4); andMMI-0500 (SEQ ID NO: 5) inhibited in vitro more than 65% of the kinaseactivities of Mitogen-Activated Protein Kinase-Activated Protein Kinase2 (MK2), Mitogen-Activated Protein Kinase-Activated Protein Kinase 3(MK3), Calcium/Calmodulin-Dependent Protein Kinase I (CaMKI,serine/threonine-specific protein kinase), and BDNF/NT-3 growth factorsreceptor (TrkB, tyrosine kinase).

TABLE 1 Kinase Profiling Assay MMI-0100 MMI-0200 MMI-0300 MMI-0400MMI-0500 (SEQ ID NO: 1) (SEQ ID NO: (SEQ ID NO: (SEQ ID NO: (SEQ ID NO:(100 μM) 19) (100 μM) 3) (100 μM) 4) (100 μM) 7) (100 μM) Abl (h) 136107 69 84 16 Abl (H396P) (h) 130 121 101 105 51 Abl (M351T) (h) 128 11990 121 61 Abl (Q252H) (h) 105 107 82 98 40 Abl(T315I) (h) 98 108 97 10516 Abl(Y253F) (h) 104 102 86 78 29 ACK1 (h) 106 97 104 95 64 ALK (h) 11895 19 16 12 ALK4 (h) 124 152 140 130 81 Arg (h) 89 82 72 84 22 AMPKα1(h) 107 108 71 87 35 AMPKα2 (h) 121 88 54 58 9 ARK5 (h) 108 93 78 69 20ASK1 (h) 100 101 80 69 −4 Aurora-A (h) 120 107 92 119 110 Aurora-B (h)94 166 128 150 5 Axl (h) 81 99 52 41 12 Bmx (h) 62 76 N/D 26 45 BRK (h)70 127 35 18 41 BrSK1 (h) 100 93 67 76 72 BrSK2 (h) 129 102 83 86 84 BTK(h) 112 100 102 94 18 BTK(R28H) (h) 91 104 74 24 10 CaMKI (h) 13 21 1 0−1 CaMKIIβ (h) 58 53 2 11 3 CaMKIIγ (h) 106 94 5 3 3 CaMKIδ (h) 59 47 1017 0 CaMKIIδ (h) 89 2 1 2 1 CaMKIV (h) 87 71 17 18 −1 CDK1/cyclinB (h)96 115 73 74 57 CDK2/cyclinA (h) 97 114 86 92 87 CDK2/cyclinE (h) 106112 94 83 19 CDK3/cyclinE (h) 106 104 94 92 8 CDK5/p25 (h) 114 97 89 9266 CDK5/p35 (h) 94 92 79 76 59 CDK6/cyclinD3 (h) 103 100 86 85 23CDK7/cyclinH/MAT1 (h) 89 67 65 47 15 CDK9/cyclin T1 (h) 228 103 91 235 6CHK1 (h) 97 115 91 87 65 CHK2 (h) 104 105 66 54 13 CHK2(I157T) (h) 97 8543 41 3 CHK2(R145W) (h) 97 81 33 31 3 CK1γ1 (h) 110 98 111 116 109 CK1γ2(h) 119 104 123 114 119 CK1γ3 (h) 105 96 125 115 114 CK1δ (h) 115 92 9293 78 CK2 (h) 90 83 90 101 93 CK2α2 (h) 104 88 105 96 103 CLK2 (h) 88 97103 116 116 CLK3 (h) 108 76 61 84 76 cKit (h) 95 110 53 43 45cKit(D816V) (h) 117 118 60 35 30 cKit(D816H) (h) 79 106 126 143 194cKit(V560G) (h) 94 115 102 124 198 cKit(V654A) (h) 69 113 134 150 223CSK (h) 70 33 49 16 2 c-RAF (h) 97 115 107 102 19 cSRC (h) 70 32 26 1430 DAPK1 (h) 97 113 46 36 0 DAPK2 (h) 41 92 32 16 3 DCAMKL2 (h) 146 13181 70 56 DDR2 (h) 105 104 94 95 79 DMPK (h) 60 66 59 54 12 DRAK1 (h) 4734 14 14 8 DYRK2 (h) 99 142 155 195 127 eEF-2K (h) 113 136 91 43 43 EGFR(h) 95 83 21 16 −1 EGFR(L858R) (h) 76 120 N/D 52 26 EGFR(L861Q) (h) 5374 25 22 15 EGFR(T790M) (h) 106 113 100 106 70 EGFR(T790M, L858R) (h) 93108 85 78 53 EphA1 (h) 114 136 73 61 40 EphA2 (h) 58 95 31 17 N/D EphA3(h) 107 117 6 12 33 EphA4 (h) 110 127 88 65 48 EphA5 (h) 110 123 18 2442 EphA7 (h) 193 220 159 222 189 EphA8 (h) 181 133 93 146 337 EphB2 (h)68 128 18 22 70 EphB1 (h) 99 95 44 58 37 EphB3 (h) 109 128 62 47 79EphB4 (h) 62 131 44 28 38 ErbB4 (h) 73 82 40 0 2 FAK (h) 98 110 111 9694 Fer (h) 117 101 130 108 196 Fes (h) 44 74 20 16 23 FGFR1 (h) 120 9755 59 18 FGFR1(V561M) (h) 108 72 74 74 113 FGFR2 (h) 49 73 14 18 12FGFR2(N549H) (h) 95 104 116 112 105 FGFR3 (h) 73 208 102 0 10 FGFR4 (h)67 75 28 19 3 Fgr (h) 54 71 60 47 109 Flt1 (h) 109 96 69 48 27Flt3(D835Y) (h) 120 115 80 71 65 Flt3 (h) 104 99 84 18 17 Flt4 (h) 135105 83 89 73 Fms (h) 89 92 45 37 14 Fms(Y969C) (h) 126 88 72 91 N/D Fyn(h) 71 75 74 54 83 GCK (h) 98 99 70 66 30 GRK5 (h) 117 135 136 131 116GRK6 (h) 131 132 147 141 174 GRK7 (h) 111 124 122 100 93 GSK3α (h) 183119 157 164 175 GSK3β (h) 113 132 205 202 238 Haspin (h) 127 71 48 36 25Hck (h) 354 107 72 72 78 Hck (h) activated 58 100 82 81 67 HIPK1 (h) 94115 74 91 47 HIPK2 (h) 98 102 73 90 38 HIPK3 (h) 105 105 93 105 85IGF-1R (h) 102 49 119 90 117 IGF-1R (h), activated 126 94 80 77 45 IKKα(h) 108 104 93 87 50 IKKβ (h) 105 109 84 84 71 IR (h) 112 90 96 85 95 IR(h), activated 127 105 79 59 90 IRR (h) 85 69 8 8 10 IRAK1 (h) 97 101 9593 5 IRAK4 (h) 100 110 59 59 3 Itk (h) 99 98 77 63 7 JAK2 (h) 89 131 133119 49 JAK3 (h) 150 117 121 122 95 JNK1α1 (h) 91 106 97 98 109 JNK2α2(h) 114 109 98 96 81 JNK3 (h) 104 90 89 70 171 KDR (h) 100 110 101 94 15Lck (h) 346 113 −2 228 359 Lck (h) activated 106 90 243 216 76 LIMK1 (h)103 109 88 92 87 LKB1 (h) 111 99 101 89 51 LOK (h) 37 67 37 18 7 Lyn (h)113 98 69 3 31 MAPK1 (h) 108 97 107 100 102 MAPK2 (h) 98 105 98 93 60MAPKAP-K2 (h) 19 35 5 5 9 MAPKAP-K3 (h) 27 39 3 7 9 MEK1 (h) 86 116 7777 21 MARK1 (h) 109 102 132 120 110 MELK (h) 74 59 16 17 0 Mer (h) 47 9052 50 17 Met (h) 104 71 65 62 27 Met(D1246H) (h) 99 139 125 68 150Met(D1246N) (h) 114 149 82 31 90 Met(M1268T) (h) 114 143 255 265 239Met(Y1248C) (h) 77 141 84 36 73 Met(Y1248D) (h) 87 118 102 31 218Met(Y1248H) (h) 88 153 117 63 126 MINK (h) 96 103 48 52 5 MKK6 (h) 74 9848 44 18 MKK7β (h) 137 117 100 94 102 MLCK (h) 85 103 2 1 0 MLK1 (h) 7784 40 33 43 Mnk2 (h) 94 106 89 86 6 MRCKα (h) 98 103 104 97 5 MRCKβ (h)103 102 83 71 −10 MSK1 (h) 52 50 32 28 8 MSK2 (h) 105 88 56 52 14 MSSK1(h) 82 100 77 75 22 MST1 (h) 85 72 14 6 3 MST2 (h) 98 104 19 11 2 MST3(h) 104 95 45 36 4 mTOR (h) 102 110 91 93 135 mTOR/FKBP12 (h) 117 118145 125 140 MuSK (h) 85 106 93 93 27 NEK2 (h) 102 97 78 61 0 NEK3 (h)100 100 92 85 20 NEK6 (h) 109 98 82 85 49 NEK7 (h) 97 96 84 87 89 NEK11(h) 102 95 53 33 2 NLK (h) 100 106 87 90 19 p70S6K (h) 89 84 35 33 3PAK2 (h) 71 69 65 59 44 PAK4 (h) 92 98 94 89 86 PAK3 (h) N/D 50 140 121102 PAK5 (h) 97 100 110 117 125 PAK6 (h) 121 105 104 100 107 PAR-1Bα (h)62 110 113 109 97 PASK (h) 81 60 29 28 9 PDGFRα (h) 104 108 65 40 40PDGFRα (D842V) (h) 103 107 114 118 170 PDGFRα (V561D) (h) 58 106 82 100146 PDGFRβ (h) 116 137 81 53 40 PDK1 (h) 144 143 135 159 178 PhKγ2 (h)62 86 46 38 16 Pim-1 (h) 44 18 8 7 0 Pim-2 (h) 117 74 76 92 46 Pim-3 (h)98 94 80 80 37 PKA (h) 138 110 119 119 118 PKBα (h) 140 110 57 67 30PKBβ (h) 284 250 84 98 21 PKBγ (h) 105 103 20 41 20 PKCα (h) 94 100 8986 3 PKCβI (h) 88 98 78 78 1 PKCβII (h) 102 100 82 75 3 PKCγ (h) 94 10189 79 6 PKCδ (h) 100 101 101 90 61 PKCε (h) 102 98 79 59 23 PKCη (h) 105101 103 98 45 PKCτ (h) 110 97 68 46 7 PKCμ (h) 79 73 22 14 10 PKCθ (h)102 101 88 76 62 PKCζ (h) 82 98 81 75 7 PKD2 (h) 84 78 33 25 10 PKG1α(h) 82 70 64 58 25 PKG1β (h) 71 57 50 53 24 Plk1 (h) 109 128 115 119 104Plk3 (h) 107 107 127 129 122 PRAK (h) 159 115 128 118 95 PRK2 (h) 72 7433 27 7 PrKX (h) 84 112 61 76 57 PTK5 (h) 135 108 132 129 96 Pyk2 (h)113 127 47 34 46 Ret (h) 108 96 140 145 174 Ret (V804L) (h) 113 100 7973 20 Ret(V804M) (h) 92 105 95 87 36 RIPK2 (h) 92 98 97 98 30 ROCK-I (h)99 117 79 73 17 ROCK-II (h) 102 85 74 77 2 Ron (h) 117 120 93 79 46 Ros(h) 107 86 95 99 150 Rse (h) 109 88 88 89 63 Rsk1 (h) 86 102 46 54 34Rsk2 (h) 65 101 51 38 14 Rsk3 (h) 76 109 76 71 23 Rsk4 (h) 99 125 90 9129 SAPK2a (h) 110 107 90 85 52 SAPK2a(T106M) (h) 101 100 97 99 32 SAPK2b(h) 99 95 81 82 42 SAPK3 (h) 106 97 84 79 24 SAPK4 (h) 98 106 96 91 48SGK (h) 128 115 48 54 2 SGK2 (h) 103 119 56 98 −1 SGK3 (h) 95 58 10 8 −3SIK (h) 113 102 66 68 40 Snk (h) 94 109 114 131 122 Src(1-530) (h) 95 7523 19 21 Src(T341M) (h) 98 56 70 76 59 SRPK1 (h) 69 93 90 96 80 SRPK2(h) 92 100 106 97 80 STK33 (h) 99 98 45 52 16 Syk (h) 45 36 24 9 5 TAK1(h) 116 124 122 177 N/D TAO1 (h) 99 105 82 73 24 TAO2 (h) 95 93 70 74 15TAO3 (h) 45 102 77 67 12 TBK1 (h) 106 98 37 39 16 Tec (h) activated 10077 56 29 33 Tie2 (h) 28 53 26 21 22 Tie2(R849W) (h) 102 89 117 108 106Tie2(Y897S) (h) 99 85 83 87 80 TLK2 (h) 113 129 114 151 133 TrkA (h) 74N/D 25 17 24 TrkB (h) 4 7 5 8 12 TSSK1 (h) 99 98 79 79 46 TSSK2 (h) 10791 98 94 92 Txk (h) 87 98 48 37 10 ULK2 (h) 123 132 122 131 124 ULK3 (h)142 164 167 147 177 WNK2 (h) 95 94 64 54 8 WNK3 (h) 100 97 77 74 9 VRK2(h) 112 109 161 185 169 Yes (h) 49 93 67 14 N/D ZAP-70 (h) 79 58 75 33 1ZIPK (h) 80 67 28 13 1 N/D: % activity could not be determined as theduplicates.

YARAAARQARAKALARQLGVAA (SEQ ID NO: 1)

YARAAARQARAKALNRQLGVA (SEQ ID NO: 19)

FAKLAARLYRKALARQLGVAA (SEQ ID NO: 3)

KAFAKLAARLYRKALARQLGVAA (SEQ ID NO: 4)

HRRIKAWLKKIKALARQLGVAA (SEQ ID NO: 7)

Example 2 Formulation of MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)and its Functional Equivalents

According to some embodiments, MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ IDNO: 1) and its functional equivalents are formulated as a lyophilizedpowder via spray drying, micronization (e.g., jet-milling), or as liquidformulation for nebulization.

Spray Drying

In some embodiments, spray drying is utilized for preparing MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) and its functional equivalents inconsideration of the following factors:

(a) proteins and peptides are prone to denaturation—that is, disruptionto tertiary and sometimes secondary structures;

(b) the denaturation can be reversible or irreversible and can be causedby a variety of conditions such as increase in temperature, decrease intemperature, extremes of pH, addition of solvents, pressure, and sheardenaturation (this applies to micronization);

(c) denatured proteins are less active and not therapeutic, sometimescompletely inactive;

(d) spray drying is able to turn these amorphous, large molecules intodiscrete spherical particles with a specified particle sizedistribution, controlled by processing parameters; the spray driedparticles can be very spherical, donut-shaped and are typically hollow,meaning that particles >5 μm can still be respirable but be resistant toclearance mechanisms in the lungs; and

(e) Spray drying, with or without excipients, generally improves thestability of proteins.

A lyophilized formulation of MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ IDNO: 1) and its functional equivalents is assessed for potential synergywith the spray drying process (e.g., matching of optimal moisture levelsbuffer concentrations/pH, excipient selection and the like) to ensureprotection of peptide stability.

Initial spray drying runs target mutually agreed acceptance criteriawith the aim of defining process parameters for the spray dryingoperation. For an inhalation product, particle size is considered animportant criterion. For alveolar deposition in the region of interest(Type IIs), Mass Median Aerodynamic Diameters (MMADs) of 1-5 micronsgenerally are accepted as being suitable for peripheral deposition inthe alveolar spaces (Heyder, J. Proc Am Thorac Soc, 1(4): 315-320, 2004,incorporated by reference herein). Other studies have suggested thatMMADs of 1-3 microns are a desirable starting target particle sizes forthe spray drying process. Since the likely biospace target for MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) is the alveolar region, a MMAD inthe about 2 micron range is targeted initially to ensure deposition intothe alveolar space.

Acceptance criteria include, but are not limited to, (1) particle size(i.e., D₉₀ of about 2 μm); (2) moisture levels (i.e., moisture less than3% w/w); (3) powder density; and (4) surface appearance (spherical,rough, toroidal).

A process design experiment then is conducted to optimize spray dryingprocess parameters, including, for example, but not limited to, (1)inlet pressure and drying temperature; (2) feedstock concentration andfederate; and (3) peptide/excipient ratio (excipients are, for example,buffer salts and a monosaccharide)

Example 3 Production of Batches of MMI-0100 (YARAAARQARAKALARQLGVAA; SEQID NO: 1) for Continued Aerosol Performance Assessment

2-3 spray drying runs at the defined process parameters described aboveare performed to generate material for aerosol performance assessment.

Spray-dried powders are well suited for delivery from an inhaler, forexample, without limitation, a MicroDose inhaler. MicroDose routinelyachieves both high emitted dose, and high fine particle fraction anddose with this formulation approach, both for neat as well asco-spray-dried blends. Exemplary aerosol performances for spray-driedinsulin are shown in FIGS. 1 and 2.

Although dry micronization is a preferred powder production method forsmall molecules for pulmonary delivery, in contrast to spray-drying, itis a stressful method, which uses high shear forces. Because use of highshear forces may lead to fracturing of proteins and peptides, drymicronization is not routinely used for large molecules. In addition, ifdose sizes are small, bulking agents are needed to improve theflowability and allow accurate measurement of the powders in fillingoperations. The primary excipient, and one of the only excipientsapproved for pulmonary delivery for this purpose is lactose, may need tobe tested for chemical compatibility with MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or its functional equivalents aslactose is incompatible with certain peptides.

The micronization process is fairly straightforward and well known inthe art. Briefly, lyophilized dry powder of MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) and its functional equivalentsare run through milling stages until the prescribed target particle sizedistribution (i.e., MMAD, D10, D50, D90) is achieved. This neatmicronized powder is tested for potency to ensure its activity postmicronization, optimized for delivery from the inhaler, and its chemicaland physical stability tested in the primary (heat sealed blister)packaging. The neat powder then is blended with a prioritized selectionof approved pulmonary lactose grades to a target, tested for blendhomogeneity, and run through the same inhaler optimization and stabilitytesting.

MicroDose Dry Powder Inhaler (DPI)

According to some embodiment, MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ IDNO: 1) and its functional equivalents can be administered using a drypowder inhaler (DPI). For example, the MicroDose dry powder inhaler(DPI) has an ‘active’ piezo driven aerosol generator that is breathactuated and achieves high efficiency of lung delivery independent ofthe patient's inhalation flow rate and volume. Unlike ‘passive’ DPIsthat require a strong and forceful inhalation on the order of 40-60liters per minute (LPM) flow for effective lung delivery, there is nobreathing maneuver required for the MicroDose DPI, as it can delivereffectively over a very broad range of flow rates from as low as 10 LPMup to 90 LPM flow, with equivalent performance (see performance examplesin FIGS. 3 and 4).

According to some other embodiments, MMI-0100 (YARAAARQARAKALARQLGVAA;SEQ ID NO: 1) and its functional equivalents can be administered using atidal breathing application, such as a ‘dry powder nebulizer’ (DPN). TheDPN delivers dry powder doses synchronized to inhalation tidal breathingwith triggering as low as 2 litter per minute (LPM), expected peak flowsof between 5 and 15 LPM and tidal volumes as low as 30 ml, which aremuch more challenging conditions than are expected with adult IPFpatients. This new DPN has successfully completed its second clinicaltrial in adults, with completion of its first study in November, 2011.These results are accessible via internet on the world wide web (www) atthe URL“clinicaltrials.gov/ct2/show/NCT01489306?spons=Microdose&rank=1.”

The MicroDose electronic inhaler system is an extremely flexibleformulation, can accurately and efficiently deliver both formulationmodalities above with particularly high efficiency with spray dried drugproducts, and has shown this performance capability with over 30 smalland large molecules. Spray-dried insulin in the primary packaging, forexample, can last at least 18 months. Examples of delivery performancefor both spray-dried peptides and micronized small molecules are shownin FIGS. 5-8.

As for the effect of the dry powder formulation on pulmonary membranes,e.g., sensitization, dry powder delivery, especially at low powder loads(<4-5 mg), is unlikely to affect pulmonary membranes or causesensitization (cough, etc.) unless this is an intrinsic property of theactive molecule (which we have not observed in animal studies).Excipients that have already been pulmonarily approved with excellentpulmonary biocompatibility are selected, and are present in very lowquantities (i.e., low mg range). For instance, mannitol at lowquantities is not likely to have an effect.

Liquid Nebulization

Alternatively, MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or itsfunctional equivalents can be delivered via liquid nebulization.Previous preclinical studies have shown that MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) can be delivered to rodents viaan AeroGen® nebulizer system adapted for animal use.

In order to specifically address MMI-0100's (YARAAARQARAKALARQLGVAA; SEQID NO: 1) ability to be delivered to positively impact compromised lung,in the bleomycin animal model efficacy experiments, a solution ofMMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) is effectivelyaerosolized. Local lung MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)administration is achieved via a rodent nebulizer device designed andmanufactured by Aerogen® (www.aerogen.com). The Aeroneb® Lab MicropumpNebulizer uses a high-efficiency aerosolization technology for use inpreclinical aerosol research and inhalation studies, providing avaluable link between preclinical and clinical product development. Theflow-rate is >0.3 ml/min, and is designed to deliver 2 mm-sizedparticles, with distribution into deepest alveoli. Efficacy of nebulizedMMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) and cellular uptakethroughout the lung has been demonstrated in the bleomycin mouse modelof pulmonary fibrosis (see FIG. 16). Localized, clinically-relevantinhaled administration is as effective as conventional systemicinjections in attenuating MK2 activation

Example 4 The Level of Activated MK2 is Increased in Fibrotic Lesions ofPatient Lungs with Idiopathic Pulmonary Fibrosis (IPF)

Mitogen-activated protein kinase (MAPK)-activated protein kinase 2 (MK2)is activated upon stress by p38MAPK-α and β. These two isoforms ofp38MAPK bind to a basic docking motif in the carboxy terminus of MK2,which subsequently phosphorylate its regulatory sites. As a result ofactivation, MK2 is exported from the nucleus to the cytoplasm andco-transports active p38 MAPK to this compartment. MK2 stabilizesp38MAPK localization and is essential for differentiation, migration andcytokine production (Kotlyarov, A., Mol Cell Biol. 22(13): 4827-4835,2002).

Therefore, in order to examine whether the p38MAPK-MK2 signaling pathwayis activated in the lungs affected by IPF, lung sections obtained fromnormal and IPF patients were stained with a phospho-specific antibodyagainst an activated form of MK2 (anti-phospho-Thr³³⁴-MAPKAPK2). Normallung and IPF lung tissues were immunostained using DAB and nucleus wascounterstained with Hematoxylin. As shown in FIG. 9, increasedexpression of activated MK2 was observed cells in the fibrotic foci fromlung tissue explants derived from patients with IPF as compared withnormal lung biopsy tissue (left). These results suggest that fibrosisformation in the lungs of IPF patients is characterized by aberrantactivation of the p38MAPK-MK2 signaling pathway.

Example 5 Nebulized and Systemic Administration of MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) Protects AgainstBleomycin-Induced Lung Fibrosis in Mice

One of the hallmarks of idiopathic pulmonary fibrosis (IPF) is theactivation of mesenchymal cells and exuberant deposition of matrix,specifically collagen. The resultant accumulation of collagen in thelung can be measured both by histological and biochemical techniques,most notably via accumulation of hydroxyproline, which is almost totallyderived from collagen in the lung and thus serves as a surrogate forwhole lung collagen content (Umezawa H. et al., Cancer, 20(5):891-895,1967).

Therefore, the therapeutic efficacy of the MMI-0100 peptide(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) on treating pulmonary fibrosiswas assessed using a mouse model of bleomycin-induced pulmonary fibrosisby delivering the MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)peptide systemically (intraperitoneal) or locally (via nebulized dosing)during prophylaxis or pre-fibrotic stage (i.e., drug administrationbeginning at day 7 post bleomycin injury; See FIG. 10) and by measuringthe levels of collagen as indices of fibrosis in the bleomycin mouse.

Briefly, fibrotic loci in the lungs of the mice were induced bydelivering intratracheally about 0.025 U of bleomycin (dissolved in PBS)to C57BL/6 mice. In order to examine the efficacy of MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) in the treatment of thebleomycin-injured lungs in the prophylaxis/pre-fibrotic phase, a control(PBS) or MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) wasadministered daily, either intraperitoneally or via nebulization,starting at day 7 post bleomycin delivery (when inflammation subsidesand fibrotic mechanisms are activated) until day 21 post bleomycindelivery (when significant fibrosis is observed) (FIG. 10). At 21 daypost bleomycin delivery, lung tissues from the bleomycin mice treatedwith MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or a control (PBS),were isolated, fixed, embedded in paraffin, and sectioned for staining.Briefly, mice were sacrificed with a sodium pentobarbital injection (120mg/kg) and the chest cavity was opened. The right mainstem bronchus wasligated and the right lung was removed. The trachea was cannulated andthe left lung was perfused with 4% formaldehyde at 21 cm H₂O pressure.The tissue blocks then were embedded in paraffin, and 4-mm sections wereprepared, and stained with hematoxylin and eosin (H&E; for pathologicalexamination) or Masson's Trichrome (for collagen staining)

As shown in FIG. 11, the lung sections from PBS-treated mice exhibitednormal lung structures (NL) and airways (AW). In contrast, the lungsections from the bleomycin mice (at day 21) revealed narrowed airway(AW) structure with formation of fibrotic foci (FF) (upper panel;Hematoxylin & Eosin (H&E) staining) and increased accumulation ofcollagen (arrows in the lower panel; Masson's Trichrome staining) inlung tissues, which are reminiscence of those found in IPF patients.Administration of MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1),either via nebulization or intraperitoneally, however, significantlyreduced development of fibrotic loci formation (upper panel, MMI-0100(NEB) and MMI-0100 (IP)) and reduced collagen accumulation (lower panel,MMI-0100(NEB) and MMI-0100 (IP)) in the lungs of the bleomycin mice.

Next, total collagen levels in the lungs of the bleomycin-injured mice(FIG. 12) were analyzed quantitatively by computing a constantconversion factor (7.5) for collagen from hydroxyproline concentrations(Neuman R. and Logan M, J Biol Chem., 186(2):549-56, 1950, incorporatedby reference). As shown in FIG. 12, both nebulized (BLEO+NEBULIZED) andsystemic (BLEO+IP) administration of MMI-0100 (YARAAARQARAKALARQLGVAA;SEQ ID NO: 1) during the post-inflammatory/pre-fibrotic stagesignificantly decreased collagen deposition compared to the bleomycincontrol.

Example 6 Dose-Response Data of MK2 Peptide Inhibitors in the IdiopathicPulmonary Fibrosis Prevention Model

Next, the effect of increasing doses of MK2 peptide inhibitors oncollagen deposition was examined in vivo using the bleomycin mouse modelof idiopathic pulmonary fibrosis (prevention model). Briefly, C57-BL/6mice were subjected to bleomycin injury at day 0. Beginning at day 7 andcontinuing through day 21, mice were administered 25, 50 or 75 μg/kg ofMMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or MMI-0200(YARAAARQARAKALNRQLGVA; SEQ ID NO: 19) daily via intra-peritoneal (IP)injection. As shown in FIG. 13, Masson's blue trichrome stainingrevealed a decrease in collagen levels in the lung of the bleomycininjured mouse treated with MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO:1), suggesting that MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) canprotect fibrosis in the lungs due to bleomycin injury in adose-dependent manner. These data suggest that MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) retains its potential as afibroprotective compound even at higher doses.

In contrast, treatment of the bleomycin injured mice with MMI-0200(YARAAARQARAKALNRQLGVA; SEQ ID NO: 19) did not reduce, but ratherincreased the collagen deposition in the lung at the doses tested. Thisresult, however, is consistent with a previous study involving MK2knockout mice and MK2−/− mouse embryonic fibroblast (MEFs), in which allMK2 activity was ablated, which exhibited an aggravated fibrosisphenotype (Liu et al., Am J Respir Cell Mol Biol, 37: 507-517, 2007).

Without being limited by theory, these results suggest (1) that MK2inhibitory peptides of the described invention may exhibit a spectrum ofinhibitory activities against a specific group of kinases; (2) MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) and MMI-0200(YARAAARQARAKALNRQLGVA; SEQ ID NO: 19) may inhibit MK2 and other kinasesdifferentially, which, depending on the dose applied, contributes tothis spectrum of inhibitory activities; (3) that myofibroblast formationand/or migration might also be a part of the repair phase of fibrosisrather than of active damage; and (4) that a certain level of MK2activity is, therefore, necessary for that process to occur (Liu et al.,Am J Respir Cell Mol Biol, 37: 507-517, 2007).

In addition, the MK2 inhibitory peptides of the described invention werederived from the substrate binding site of MK2 downstream target HSPB1.Therefore, they can competitively inhibit the kinase activity of MK2toward HSPB1. Without being limited by theory, the differential effectsof MMI-0200 (YARAAARQARAKALNRQLGVA; SEQ ID NO: 19) on fibrosis may beattributed to its sequence differences, its homology to the HSPB1 bidingsites, its differential inhibition of MK2 kinase activity toward adistinct target protein binding site, or a combination thereof.

Example 7 Administration of MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ IDNO: 1) Effectively Blocks Systemic T-Cell Activation in IdiopathicPulmonary Fibrosis Prevention Model

Recent studies highlighted a key role for T lymphocytes inbleomycin-induced fibrosis (Wilson, M. et al., The Journal ofExperimental Medicine, 207(3): 535-552, 2010). Therefore, in order toinvestigate the functional role of splenic (pan) T cells in thebleomycin injured mice treated with MMI-0100 (YARAAARQARAKALARQLGVAA;SEQ ID NO: 1), the autologous mixed lymphocyte reaction (MLR) wasperformed as described previously (Wilkes, D. et al., Journal ofLeukocyte Biology, 64(5):578-586, 1998, incorporated by referenceherein). Specifically, the ability of C57BL/6 purifiedantigen-presenting cells to induce proliferation in C57BL/6 Tlymphocytes was examined in the assay.

C57-BL/6 mice were subjected to bleomycin injury at day 0. At day 7, themice were administered 50 μg/kg/day MMI-0100 (YARAAARQARAKALARQLGVAA;SEQ ID NO: 1) daily by intraperitoneal (IP) injection or nebulizer (NEB)until day 21. Splenic T cells were isolated and cultured alone or in thepresence of autologous antigen presenting cells (APCs from C57-BL/6mice) or stimulated with antibodies against CD3 (α-CD3) for 48 h. Thecells then were radiolabeled with triturated thymidine for 16 h andassessed for proliferation rates.

As shown in FIG. 14, T cells alone, regardless of treatment, exhibitedvery low proliferative capacity. However, when the T cells wereco-cultured with autologous antigen presenting cells (i.e., APCsisolated from C57-BL/6 mice), the proliferative capacity wassignificantly higher for bleomycin-injured mice than for control mice.Interestingly, the proliferation of T cells from bleomycin treated miceseen in the presence of antigen presenting cells was significantlyreduced by the systemic administration of MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1), but as expected, not by theinhaled mode. These data suggest the suppression of splenic T cellactivation by MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1), andindicate that the peptide MK2 inhibitor is fibro-protective.

The viability of the T cells also was confirmed by stimulating the cellswith antibodies against α-CD3, a polyclonal T cell activator. α-CD3induced robust proliferation of the cells irrespective of the treatmentgroup. The proliferative response to the polyclonal activator suggeststhat the MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) peptideinhibitor does not affect the functional property of the splenic Tcells, and that there is no toxicity with the administration of MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) at this particular dose. Inaddition, the lack of splenic T cell response to nebulized MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) suggests that little systemicdistribution occurs with this mode of peptide delivery.

Example 8 Systemic or Nebulized MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ IDNO: 1) Treatment Protects Bleomycin Injured Lungs in the Post-FibroticPhase

The classic bleomycin model, as depicted in FIG. 10, has been usedwidely in the literature in the pre-fibrotic stage to test efficacy ofany intervention. Since both nebulized and systemic administration ofthe MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) significantlyprotected the lung from bleomycin-induced fibrosis, the effect ofsystemic (intraperitoneal) or local (nebulized) administration ofMMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) in the treatment ofbleomycin injured lungs was examined further at the post-fibrotic stage,with drug intervention being begun at day 14, a time point when thelungs are significantly fibrosed (FIG. 15) (Pottier, N. et al., AmericanJournal of Respiratory and Critical Care Medicine, 176(11): 1098-1107,2007, incorporated by reference herein). The rescuing of scarred lungsthat is shown in this model is clinically relevant, given that lungs ofIPF patients already are scarred at the time of diagnosis.

More specifically, fibrotic loci in the lungs were induced by deliveringintratracheally about 0.025 U of bleomycin (dissolved in PBS) to C57BL/6mice. In order to examine the efficacy of MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) in the treatment ofbleomycin-injured lungs in the post-fibrotic phase, PBS (control) orMMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) was administered to themice either intraperitoneally or via nebulization daily starting at day14 post bleomycin delivery until day 28 post bleomycin delivery. At 28day post bleomycin delivery, the lung tissues of the bleomycin micetreated with MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or acontrol (PBS), were isolated, fixed, embedded in paraffin, and sectionedfor staining. Mice were sacrificed with a sodium pentobarbital injection(120 mg/kg) and the chest cavity was opened. The right mainstem bronchuswas ligated and the right lung is removed. The trachea was cannulatedand the left lung was perfused with 4% formaldehyde at 21 cm H₂Opressure. The tissue blocks then were embedded in paraffin, and 4-mmsections were prepared, and stained with hematoxylin and eosin (H&E; forpathological examination) or Masson's Trichrome (for collagen staining)

As shown in FIG. 16, regardless of the mode of drug administration,i.e., whether intraperitoneally delivered or locally applied to thelung, MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) treatment“rescued” severely scarred lungs. Histological assessment was employedto examine lung architecture (Hematoxylin & Eosin (H&E) staining, toppanel) and collagen distribution (Masson's blue trichrome staining,bottom panel). The histochemistry results show that whilebleomycin-injured lungs are severely scarred, MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)-treated mice have a clearer lungparenchyma.

Next, collagen deposition was determined quantitatively by using thestandard hydroxyproline assay with whole left lung. Total collagen(soluble and insoluble) deposition was assessed by analyzinghydroxyproline concentrations in murine lungs day 28 post bleomycininjury. MMI-0100 (YARAAARQARAKALARQLGVAA (SEQ ID NO: 1)) wasadministered at the dose of 50 μg/kg/day by intra-peritoneal injection(IP) or nebulizer (NEB) beginning at day 14 post bleomycin injury.

As shown in FIG. 17, MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)treatment significantly arrested further progression of collagendeposition, as compared to baseline, at 28 days post-bleomycin injuryand the onset of MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)treatment. This is significant because while current literature showseffective prophylaxis in drug development, when IPF patients arediagnosed, there is pre-existing fibrosis. These results also suggestthat MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) has the potentialto effectively halt or slow further progression of the disease andimprove quality of life; and that the MMI-0100 (YARAAARQARAKALARQLGVAA;SEQ ID NO: 1) peptide, if used at a higher dose and/or for a longertreatment period, may result in even greater improvement in lunghistology and physiology, and diminished fibrosis.

Example 9 Either Systemic or Local Administration of MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) is Correlated with ReducedActivated MK2 in the Bleomycin Mouse Model of Idiopathic PulmonaryFibrosis

As discussed above, one of the principal targets of MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) and its functional equivalents inthe lung is MK2 kinase, which elicits inflammatory and fibroticresponses in the affected lungs. Therefore, in order to further validatethe effects of MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) in vivo,levels of activated MK2 (phospho-Thr³³⁴-MAPKAPK2) were examined inuntreated bleomycin injured mice as well as in MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)-treated mice.

Briefly, C57-BL/6 mice were subjected to bleomycin injury at day 0. Atday 14, the mice were administered 50 μg/kg of MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) daily by intraperitoneal (IP)injection or nebulizer (NEB) until day 28 post bleomycin injury.Formalin-fixed lung tissue sections were immunostained againstphospho-Thr³³⁴ MK2. Control staining was with biotinylated secondary IgGantibody. Streptavidin-conjugated horseradish peroxidase was used with3,3′-diaminobenzidene as substrate and nuclei was counterstained withhematoxylin. Whereas the bleomycin mice showed a visible increase inactivated MK2 presence (dark nodules) if left untreated, mostparticularly in areas of significant collagen deposition, mice treatedwith MMI-0100 exhibited activated MK2 presence similar to normal tissue,with such distribution concentrated in peri-airway and blood vesselregions.

As shown in FIG. 18, regardless of the mode of delivery, i.e., eithersystemic or local administration, in contrast to the control,administration of nebulized or intraperitoneal MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) was associated with decreasedphospho-Thr³³⁴-MAPKAPK2 staining (activated form of MK2) in thebleomycin mouse model.

Example 10 MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) DownregulatesInflammatory Cytokines in Idiopathic Pulmonary Fibrosis Treatment Model

One potential mechanism by which MMI-0100 (YARAAARQARAKALARQLGVAA; SEQID NO: 1) can inhibit fibrosis formation in the lung is by decreasinglocal concentrations of pro-inflammatory cytokines, and therebydeterring recruitment of monocytes and aberrant extracellular remodelingby macrophages in the lung (e.g., increase in collagen deposition,increase in cell adhesion and migration, decrease in matrixdegradation). To explore this possibility, the ability of the MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) peptide to inhibit production ofpro-inflammatory cytokines was examined by measuring changes ininterleukin-6 (IL-6) and Tumor Necrosis Factor-alpha (TNF-α) levels upontreatment with MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)intraperitoneally or via nebulization.

Interleukin-6 (IL-6) is a multifunctional cytokine whose major actionsinclude enhancement of immunoglobulin synthesis, activation of T cells,and modulation of acute-phase protein synthesis. Many different types ofcells are known to produce IL-6, including monocytes, macrophages,endothelial cells, and fibroblasts, and expression of the IL-6 gene inthese cells is regulated by a variety of inducers. Interleukin-1β(IL-1β) and tumor necrosis factor (TNF-α) are two key known inducers ofIL-6 gene expression. Other inducers include activators of proteinkinase C, calcium inophore A23187, and various agents causing elevationof intracellular cyclic AMP (cAMP) levels.

Tumor necrosis factor (TNF, also referred as TNF-α) is a cytokineinvolved in systemic inflammation and is a member of a group ofcytokines that stimulate the acute phase reaction. Studies have shownthat TNF-α induces expression of IL-6 via three distinct signalingpathways inside the cell, i.e., 1) NF-κB pathway 2) MAPK pathway, and 3)death signaling pathway.

As shown in FIG. 20, administration of either intraperitoneal(BLEO+MMI-0100 (IP)) or nebulized (BLEO+MMI-0100 (NEB)) MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) significantly reduced the plasmalevel of both TNF-α (A, upper panel) and IL-6 (B, lower panel) in thebleomycin mouse model of idiopathic pulmonary fibrosis.

Example 11 Either Systemic or Local Administration of MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) Effectively Blocks MyofibroblastActivation Accumulation in Murine Lung that is Significantly Scarred Dueto Bleomycin-Injury

The hallmark of idiopathic pulmonary fibrosis (IPF) is the accumulationof myofibroblasts at fibrotic lesions and expression of abundantalpha-smooth muscle actin (α-SMA), a marker for myofibroblastactivation. Furthermore, activated myofibroblasts are in partresponsible for rigidity of the lung parenchyma and aggravation of lungfunction.

Therefore, the expression level of α-SMA in bleomycin-injured lungs wasassessed in the lungs of bleomycin-injured mice treated with MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1), either systemically (viaintraperitoneal administration) or locally (via nebulization). As shownin FIG. 21, the level of α-SMA was significantly attenuated in MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)-treated lungs compared to thelevel of α-SMA in untreated bleomycin-injured lungs.

Example 12 Dose Response Studies of MMI-0100 (YARAAARQARAKALARQLGVAA;SEQ ID NO: 1) in Modulating TGF-β1-Induced Myofibroblast Activation InVitro

The major hallmarks of idiopathic pulmonary fibrosis (IPF) are thepresence of atypical and apoptotic epithelial cells, along withactivated myofibroblasts that secrete exuberant amounts of matrixproteins including collagens, fibronectin and matrix metalloproteinases(Horowitz, J and Thannickal, V., Treatments in Respiratory Medicine,5(5):325-42, 2006). Under normal wound healing processes, a provisionalmatrix is formed by the myofibroblasts as a temporary scaffolding.Contraction of the provisional matrix results in subsequentre-epithelialization and eventual wound healing. However, when activatedmyofibroblasts are resistant to apoptosis, the resultant exuberantcollagen deposition leads to stabilization of the matrix (Tomasek, J. etal., Nature Reviews Molecular Cell Biology, 3(5): 349-63, 2002). Theend-result of unchecked myofibroblast proliferation, activation andresistance to apoptosis results in fibrotic lesions with stabilizedmatrix due to collagen deposition and thus eventual distortion of lungarchitecture (Yamashita, C. et al., The American Journal of Pathology,179(4): 1733-45, 2011).

Therefore, since fibroblasts are the key cells involved in scarformation, the effect of MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)on myofibroblast activation was assessed by examining the protein levelsof α-smooth muscle actin (α-SMA) and fibronectin in cultured human fetallung fibroblasts (IMR-90 cells) treated with TGF-β. As shown in FIGS. 22and 23, MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) effectivelyprevented myofibroblast activation induced by TGF-β in a dose-dependentmanner, as shown by decreases in the levels of both α-smooth muscleactin (α-SMA) (FIG. 22) and fibronectin (FIG. 23).

In contrast, MMI-0200 (YARAAARQARAKALNRQLGVA; SEQ ID NO: 19) at thedoses tested did not affect the TGF-β-mediated myofibroblast activation,as indicated by no changes in the protein level of myofibroblastactivation markers α-smooth muscle actin (FIG. 21) and fibronectin (FIG.23). Without being limited by theory, these results suggest (1) that MK2inhibitory peptides of the described invention may exhibit a spectrum ofinhibitory activities against a specific group of kinases; (2) thatMMI-0100 (SEQ ID NO: 1) and MMI-0200 (SEQ ID NO: 19) may inhibit MK2 andother kinases differentially, which, depending on the dose applied,contributes to this spectrum of inhibitory activities; (3) that theremight be compensatory pathways that regulate α-smooth muscle actin; (4)that myofibroblast formation and/or migration might also be a part ofthe repair phase of fibrosis rather than of active damage; and (5) thata certain level of MK2 activity is, therefore, necessary for thatprocess to occur (Liu et al., Am J Respir Cell Mol Biol, 37: 507-517,2007).

In addition, the MK2 inhibitory peptides of the described invention werederived from the substrate binding site of MK2 downstream target HSPB1.Therefore, they can competitively inhibit the kinase activity of MK2toward HSPB1. Without being limited by theory, the differential effectsof MMI-0200 (SEQ ID NO: 19) on fibrosis may be attributed to itssequence differences, its homology to the HSPB1 biding sites, and itsdifferential inhibition of MK2 kinase activity toward a distinct targetprotein binding site.

Example 12 Efficacy of MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)and MMI-0100 in Combination with an Antiviral, Agent, e.g., Sofosbuvir(Sovaldi®), on Liver Fibrosis in a Murine Liver Fibrosis Model

The most commonly used approach to induce experimental liverinflammation and fibrinogenesis is the periodic administration of carbontetrachloride (CCl₄) in mice (Liedtke C, et al., Fibrogenesis & TissueRepair, 2013; 6:19). The CCl₄ model resembles all the importantproperties of human liver fibrosis, including inflammation,regeneration, fiber formation and potentially fibrosis regression(Liedtke C, et al., Fibrogenesis & Tissue Repair, 2013; 6:19; HeindryckxF, et al., Int. J. Exp. Pathol., 2009; 90: 367-386; Iredale J P, et al.,J. Clin. Invest., 1998; 102: 538-549; Kisseleva T, et al., PNAS, 2012;109: 9448-9453). Most studies still rely on the CCl₄-model to inducetoxic liver fibrosis in mice due to its good comparability with theabundance of previous publications, excellent reproducibility andmoderate burden for the animals (Liedtke C, et al., Fibrogenesis &Tissue Repair, 2013; 6:19).

Pathogen free 8-10 week old female BALB/c mice can be obtained fromCharles River Laboratories (Wilmington, Mass.). The mice can be dividedinto three groups: Group 1 to receive MMI-0100 (YARAAARQARAKALARQLGVAA;SEQ ID NO: 1) or a functional equivalent; Group 2 to receive MMI-0100(YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) or a functional equivalent+one ofSofosbuvir (Sovaldi®), an HCV boosted protease inhibitor (ABT-450,AbbVie), a nonnucleoside NS5B inhibitor (dasabuvir, ABT-333, AbbVie), anNS5a inhibitor (ombitasvir, ABT-267, AbbVie), ABT-450/r (ABT-450 withritonavir), ABT-450 co-formulated with ABT-267, ABT-450 formulated withsofosbuvir, ribavirin, or a combination. Group 3 to receive phosphatebuffer saline (PBS) (Control Group). Mice can be injectedintraperitoneally two to three times per week with CCl₄ (Sigma-Aldrich,St. Louis, Mo.) (0.5 to 2 mL/kg body weight diluted in corn oil) for 4to 6 weeks. At the time of CCl₄ injection, Group 1 mice can beadministered MMI-0100 or a functional equivalent intraperitoneally(e.g., 3, 30, 100, 300 ng/kg) in 100 μL PBS; Group 2 mice can beadministered MMI-0100 or a functional equivalent+an antiviral agentintraperitoneally (e.g., 3, 30, 100, 300 ng/kg) in 100 μL PBS; and Group3 mice can be administered 100 μL PBS intraperitoneally. Samples can becollected throughout the duration of the study (e.g., blood samples canbe collected from tail veins immediately preceding each intraperitonealinjection). At the end of the study, mice can be anaesthetized and bloodand liver tissue samples can be collected prior to sacrifice in order todetermine the effect of MMI-0100 or a functional equivalent and MMI-0100or a functional equivalent+an antiviral agent on liver fibrosis.

For example, liver function can be determined by measuring serum levelsof alanine aminotransferase (ALT), aspartate aminotransferase (AST),gamma-glutamyl transferase (GGT) and albumin using commerciallyavailable kits according to manufacturer's instructions (e.g.,Sigma-Aldrich, St. Louis, Mo.).

The level of liver fibrosis can be evaluated in liver tissue, forexample, by hydroxyproline content, histology and immunohistochemistry,and mRNA expression of profibrotic and inflammatory biomarkers.

The hydroxyproline content of a liver tissue sample can be determined inthe following manner. 100 mg of liver sample can be hydrolyzed in 6 MHCl at 110° C. for 24 hours. Next, the sample is centrifuged at 2,000rpm at 48° C. for 5 minutes. 2 mL of supernatant is collected and mixedwith 50 mL of 1% phenolphthalein and 8 N KOH to pH 7-8. A 5 mL samplecan be subjected to a spectrophotometer at 560 nm to determinehydroxyproline content.

Histology and immunohistochemistry can be performed as follows. Livertissues can be fixed in 10% neutral buffered formaldehyde and embeddedin paraffin. Paraffin sections can be stained with Masson's Trichrome(Sigma-Aldrich, St. Louis, Mo.) and periodic acid-Schiff (PAS) stain(Millipore, Billerica, Mass.) to examine collagen deposition. Specificstaining for α-smooth muscle actin (α-SMA) and collagen proteins can beperformed using anti-α-SMA and anti-collagen I and III antibodies (R&DSystems, Minneapolis, Minn.; EMD Millipore, Billerica, Mass.; and abcam,Cambridge, Mass. respectively). Microscopic images of ×100-magnificationcan be acquired for liver sections stained with Masson's trichrome,collagen 1, collagen III and α-SMA. Images can be encoded on 24-bits perpixel on three channels (red, green and blue) and quantitativeassessment of the percentage of fibrotic modification revealed byMasson's trichrome, collagen 1, collagen III and α-SMA can be performedon the images. Colored images can be processed using ImageJ (WyneRasband, National Institutes of Health, Bethesda, Md.) and Matlab (TheMathWorks Inc., Natick, Mass.) to produce maps showing only the areasstained by Masson's trichrome, collagen 1, collagen III and α-SMA. A mapcan be defined as the stained area corresponding to the fibroticcomponent(s) deposited in the ECM of the liver. The Biomarker Index ofFibrosis (BIF) can be calculated as the percentage of a biomarker's(e.g., Masson's trichrome, collagen 1, collagen III and α-SMA) map tothe whole image as described by Salazar-Montes, et al. (European Journalof Pharmacology, 2008; 595(1-3): 69-77) and Salgado, et al. (MolecularTherapy, 2000; 2: 545-551).

mRNA expression of profibrotic and inflammatory biomarkers, includingMCP-1, Collagen 1a1, Collagen 3a1, TGFβ, Fibronecin-1, α-SMA andconnective tissue growth factor-1 (CTGF-1) can be assessed by Luminex®(Life Technologies, Carlsbad, Calif.) assay according to manufacturer'sinstructions. Relative expression can be normalized to hypoxanthinephosphorbosyltransferase (HPRT).

Data can be expressed as mean±standard error of mean (SEM). Statisticalanalysis can be performed using computer software such as GraphPadPrism® (GraphPad Software, Inc., San Diego, Calif.). Treatmentdifferences between groups can be analyzed by unpaired t-Test and ANOVA.

Example 13 Efficacy of MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)on Renal Fibrosis in a Murine Renal Fibrosis Model

An increasingly used and popular animal model of renal fibrosis iscomplete unilateral ureteral obstruction (UUO) (Cho M H, Korean J.Pediatr. 2010; 53(7): 735-740). The UUO procedure has the advantage thatit mimics, in an accelerated manner, the different stages of humanobstructive nephropathy leading to tubulointerstitial fibrosis: cellularinfiltration, tubular proliferation and apoptosis,epithelial-mesenchmyal transition (EMT), (myo)fibroblast accumulation,increased extracellular matrix (ECM) deposition and tubular atrophy(Bascands J-L and Schanstra J P, Kidney Int. 2005; 68(3): 925-937).These pathological features appear rapidly (all within around 1 weekafter the UUO procedure) and are highly reproducible from one experimentto another (Bascands J-L and Schanstra J P, Kidney Int. 2005; 68(3):925-937).

Pathogen free 7-8 week old male CD-1 mice can be obtained from CharlesRiver Laboratories (Wilmington, Mass.). The mice can be divided intothree groups: Group 1 to receive MMI-0100 (YARAAARQARAKALARQLGVAA; SEQID NO: 1) or a functional equivalent; Group 2 to receive phosphatebuffer saline (PBS) (Control Group); Group 3 to undergo sham surgery(i.e., without ligation) and to receive PBS. UUO can be performed asdescribed by Yamashita et al. (Journal of the American Society ofNephrology 2004; 15(1): 91-101). Briefly, after induction of generalanesthesia by intraperitoneal injection of pentobarbital (50 mg/kg bodyweight), the abdominal cavity can be exposed via midline incision andthe right ureter can be ligated at three points with 4-0 silk. From days0 to 5, Group 1 mice can be administered MMI-0100 or a functionalequivalent intraperitoneally (e.g., 3, 30, 100, 300 ng/kg) in 100 μLPBS. Group 2 and Group 3 mice can be administered 100 μL PBSintraperitoneally. Samples can be collected throughout the duration ofthe study (e.g., blood samples can be collected from tail veinsimmediately preceding each intraperitoneal injection). On day 5, micecan be anaesthetized and blood and kidney tissue samples can becollected prior to sacrifice in order to determine the effect ofMMI-0100 or a functional equivalent on renal fibrosis. UUO can beconfirmed by observation of dilation of the pelvis and proximal ureterand collapse of the distal ureter. Sham-operated kidneys withoutligation can be used as controls.

Study endpoints can include, for example, body and kidney weights;fibrosis in obstructed kidney evaluated via histological quantitativeimage analysis of picrosirius red staining (Polysciences, Inc., catalogno. 24901-250, Warrington, Pa.) (e.g., ten images/depth/kidney obtainedand assessed in a blinded fashion using light microscopy at 200× toenable sampling of 60-70% of the renal cortical area) and quantified bya composite Collagen Volume Fraction (CVF (% total area imaged)) scoreexpressed as the average positive stain across three anatomicallydistinct (e.g., 200-250 um apart) tissue sections, or depths, from theobstructed kidney; hydroxyproline content of frozen renal corticaltissue biopsies as assessed by biochemical analyses; and mRNA expressionof profibrotic and inflammatory biomarkers such as MCP-1, Collagen 1a1,Collagen 3a1, TGF-β, Fibronectin-1, α-SMA and connective tissue growthfactor-1 (CTGF-1) assessed by Luminex® (Life Technologies, Carlsbad,Calif.) assay according to manufacturer's instructions with relativeexpression normalized to hypoxanthine phosphorbosyltransferase (HPRT).

Data can be expressed as mean±standard error of mean (SEM). Statisticalanalysis can be performed using computer software such as GraphPadPrism® (GraphPad Software, Inc., San Diego, Calif.). Treatmentdifferences between groups can be analyzed by unpaired t-Test and ANOVA.

Example 14 Efficacy of MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)on Vascular Fibrosis in Spontaneously Hypertensive Rats (SHRs)

Spontaneously hypertensive rats (SHRs) are commonly used to studyvascular fibrosis (Gao D et al., PPAR Res. 2012; 2012: 856426). For thisstudy, 8-10 week old male SHR and age-matched male Wistar Kyoto (WKY)rats can be obtained from Charles River Laboratories (Wilmington,Mass.). SHRs can receive MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1)or a functional equivalent (experimental Group 1); WKY rats (controlgroup 1) can receive MMI-0100 (YARAAARQARAKALARQLGVAA; SEQ ID NO: 1) ora functional equivalent; and SHR rats can receive vehicle (PBS) only(control group 2). Experimental Group 1 and control group 1 rats can beadministered MMI-0100 or a functional equivalent intraperitoneally(e.g., 3, 30, 100, 300 ng/kg) in 100 μL PBS; control group 2 rats canreceive vehicle only. Samples can be collected throughout the durationof the study (e.g., blood samples can be collected from tail veinsimmediately preceding each intraperitoneal injection). At the end of thestudy, rats can be anaesthetized and blood and vascular tissue samplescan be collected prior to sacrifice in order to determine the effect ofMMI-0100 or a functional equivalent on vascular fibrosis.

For example, mRNA expression of profibrotic and inflammatory biomarkers,including PPARγ MCP-1, Collagen 1a1, Collagen 3a1, TGFβ, Fibronecin-1,α-SMA and connective tissue growth factor-1 (CTGF-1) can be assessed byLuminex® (Life Technologies, Carlsbad, Calif.) assay according tomanufacturer's instructions. Relative expression can be normalized toglyceraldehyde-3-phosphate dehydrogenase (GAPDH).

Protein expression of profibrotic biomarkers including CTGF and TGF-βcan be assessed, for example, by Western blot. Briefly, protein samples(20 ug) can be resolved on 10% SDS-PAGE, transferred to polyvinylidenedifluoride membranes in a semidry system (Bio-Rad, Hercules, Calif.) andincubated with antibodies against CTGF (1:500) (Santa CruzBiotechnology, Santa Cruz, Calif.), TGF-β (1:500) (Santa CruzBiotechnology, Santa Cruz, Calif.) and β-actin (1:2000) (Santa CruzBiotechnology, Santa Cruz, Calif.). Protein can be visualized bychemiluminescence (Pierce Corp., Rockford, Ill.) and quantified using aGel Doc 2000 system (Bio-Rad).

Protein expression of profibrotic biomarkers including PARAγ andcollagen type III (Col III) can be assessed, for example, byimmunohistochemistry. Briefly, paraffin-embedded rat thoracic aortasections can be incubated with primary antibodies against PPARγ(1:300)(Upstate Inc., Chicago, Ill.) and Col III (1:250) (Upstate Inc.,Chicago, Ill.) overnight at 4° C. and then biotinylated andaffinity-purified IgG (Zymed USA) secondary antibody for 1 hr at 37° C.A streptavidin-enzyme conjugate can be sequentially added for 20 min.and samples can be incubated with substrate 3′,3′-diaminobenzidine (DAB)(Sigma-Aldrich, St. Louis, Mo.) followed by hematoxylin counterstaining(Sigma-Aldrich, St. Louis, Mo.). Quantitative analysis can be performedusing a Qwin 550 quantitative image analysis system (Leica, Germany) bymeasuring the gray scale. A negative control can includeparaffin-embedded sections not incubated with primary antibody.

Data can be expressed as mean±standard error of mean (SEM). Statisticalanalysis can be performed using computer software such as GraphPadPrism® (GraphPad Software, Inc., San Diego, Calif.). Treatmentdifferences between groups can be analyzed by unpaired t-Test and ANOVA.

While the described invention has been described with reference to thespecific embodiments thereof it should be understood by those skilled inthe art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adopt aparticular situation, material, composition of matter, process, processstep or steps, to the objective spirit and scope of the describedinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method for reducing progression of a fibrosisin liver tissue, kidney tissue or vascular tissue; treating fibroticremodeling of the liver tissue, kidney tissue or vascular tissue; orboth, in a subject in need thereof, comprising: administering to thesubject a pharmaceutical composition comprising a therapeutic amount ofa polypeptide of the amino acid sequence YARAAARQARAKALARQLGVAA (SEQ IDNO: 1) or a functional equivalent thereof selected from the groupconsisting of a polypeptide of the amino acid sequenceFAKLAARLYRKALARQLGVAA (SEQ ID NO: 3) and a polypeptide of the amino acidsequence KAFAKLAARLYRKALARQLGVAA (SEQ ID NO: 4), and a pharmaceuticallyacceptable carrier thereof, progression of the fibrosis beingcharacterized by one or more of aberrant fibroblast proliferation andextracellular matrix deposition producing remodeling in liver tissue,kidney tissue or vascular tissue, wherein the therapeutic amount of thepharmaceutical composition inhibits at least 65% of activity of MK2,MK3, CaMKI, and TrkB, with limited off-target kinase inhibition, andwherein the therapeutic amount of the polypeptide reduces progression ofthe fibrosis, treats remodeling of the liver tissue, kidney tissue orvascular tissue due to the fibrosis, or both reduces progression of thefibrosis in liver, kidney or vascular tissue and treats remodeling ofthe liver tissue, kidney tissue or vascular tissue due to the fibrosis.2. The method according to claim 1, wherein the tissue fibrosis isfurther characterized by an inflammation in the tissue.
 3. The methodaccording to claim 2, wherein the inflammation is an acute or a chronicinflammation.
 4. The method according to claim 2, wherein theinflammation is mediated by at least one cytokine selected from thegroup consisting of Tumor Necrosis Factor-alpha (TNF-α), Interleukin-6(IL-6), and Interleukin-10 (IL-1β).
 5. The method according to claim 1,wherein the aberrant fibroblast proliferation and extracellular matrixdeposition in the tissue is characterized by an aberrant activity ofMitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2) in thetissue compared to the activity of Mitogen-Activated ProteinKinase-Activated Protein Kinase 2 (MK2) in the tissue of a normalhealthy control subject.
 6. The method according to claim 1, wherein thestep of administering occurs orally, intratracheally, parenterally,intravenously, or intraperitoneally.
 7. The method according to claim 1,wherein the pharmaceutical composition further comprises at least oneadditional therapeutic agent.
 8. The method according to claim 7,wherein the additional therapeutic agent is an anti-infective agent. 9.The method according to claim 8, wherein the anti-infective agent is anantiviral agent.
 10. The method according to claim 9, wherein theantiviral agent is one or more of sofosbuvir, an HCV boosted proteaseinhibitor, a nonnucleoside NSSB inhibitor, an NS5a inhibitor, ABT-450with ritonavir, ABT-450 co-formulated with ABT-267, ABT-450 formulatedwith sofosbuvir, and ribavirin.
 11. The method according to claim 7,wherein the additional therapeutic agent is a glucocorticoid selectedfrom the group consisting of prednisone, budesonide, mometasone furoate,fluticasone propionate, fluticasone furoate, and a combination thereof.12. The method according to claim 7, wherein the additional therapeuticagent is an analgesic agent.
 13. The method according to claim 7,wherein the additional therapeutic agent is selected from the groupconsisting of a purified bovine Type V collagen, an IL-13 receptorantagonist, a protein tyrosine kinase inhibitor, an endothelial receptorantagonist, a dual endothelin receptor antagonist, a prostacyclinanalog, an anti-CTGF monoclonal antibody, an endothelin receptorantagonist, AB0024, a lysyl oxidase-like 2 (LOXL2) antibody, a c-JunN-terminal kinase (JNK) inhibitor, pirfenidone, IFN-γ1b, a humanantibody against all three TGF-β isoforms, a TGF-β activation inhibitor,a recombinant human Pentraxin-2 protein (rhPTX-2), a bispecificIL-4/IL-13 antibody, an antibody targeting integrin αvβ6,N-acetylcysteine, sildenafil, a Tumor Necrosis Factor (TNF) antagonist,and a combination thereof.
 14. The method according to claim 1, whereinthe carrier is selected from the group consisting of a controlledrelease carrier, a delayed release carrier, a sustained release carrier,and a long-term release carrier.
 15. The method according to claim 13,wherein the endothelin receptor antagonist is A-selective.
 16. Themethod according to claim 13, wherein the TNF antagonist is etanercept.